Azetidin-2-one derivatives as serine protease inhibitors

ABSTRACT

The present invention relates to new 3-guanidinoalkyl-2-azetidinones of the formula ##STR1## wherein: U and W can be the same or different and are selected from the group consisting of hydrogen and an amino protecting group; 
     n is 1 to 3; 
     X is a member selected from the group consisting of hydrogen, trialkylsilyl, arylsulfonyl, amino substituted arylsulfonyl, alkylsulfonyl, arylaminocarbonyl, alkylcarbonyl and arylcarbonyl; and 
     Y is a member selected from the group consisting of hydrogen, arylalkenyl, arylalkyl, formyl, carboxy, alkoxycarbonyl, acyloxy, arylthio, arylsulfinyl, arylsulfonyl, alkylthio, alkylsulfinyl, alkylsulfonyl, arylaminocarbonyl, the radical ##STR2## in which R is hydrogen, alkyl or arylalkyl, and the radical ##STR3##   in which m is 1 to 3 and R&#39; is hydrogen or --CO 2  R&#34; wherein R&#34; is hydrogen, alkyl or arylalkyl. 
     The novel azetidinones of the present invention exhibit anti-thrombin and anti-trypsin activities and are thus useful in controlling blood coagulation and treating pancreatitis.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. Ser. No. 07/957,233filed Oct. 6, 1992, U.S. Pat. No. 5,250,677; which is a divisional ofU.S. Ser. No. 07/833,876 filed Feb. 11, 1992, now U.S. Pat. No.5,175,283; which is a divisional of U.S. Ser. No. 07/696,641 filed onMay 7, 1991, now U.S. Pat. No. 5,110,812; which is a divisional of U.S.Ser. No. 07/533,270 filed Jun. 4, 1990, now U.S. Pat. No. 5,037,819.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel 3-guanidinoalkyl-2-azetidinones usefulfor anti-thrombin and anti-trypsin activity.

2. Description of Related Art

The traditional use of compounds containing beta-lactam structures hasbeen for antibacterial activities. However, certain beta-lactams havebeen identified as having activity against serine proteases. Compoundshaving activity against serine proteases could be used to treat certaindegenerative conditions. For example, several patents, namely U.S. PatNos. 6,680,391 (issued Jul. 14, 1987), U.S. Pat. No. 4,623,645 (issuedNov. 18, 1986), and U.S. Pat. No. 4,637,999 (issued Jan. 20, 1987),disclose substituted azetidiones which are inhibitors of human leukocyteelastase (HLE), a serine protease. Such HLE inhibitors could be used totreat degenerative conditions such as arthritis and emphesyma.

Thrombin is another serine protease which cleaves fibrinogen to fibrin.Inhibitors of the enzyme could be useful for the control of bloodcoagulation. A number of synthetic inhibitors have been discovered. Fora comprehensive review, see: Pharmakologie Synthetischem ThrombinInhibitoren, J. Hamptmann and F. Markwardt, Berlin, 1986. Untilrecently, no synthetic thrombin inhibitor which contained azetidionenucleus was reported.

A recent article in Nature [J. B. Doherty, et al., Nature, 322, 192(1982)] discloses the testing of certain cephalosporin sulfoxides forinhibiting a battery of serine proteases. Their ability to inhibitthrombin and trypsin was generally found to be much weaker than theirability to inhibit HLE.

More remote art of the present invention is found in European PatentApplication Nos. 264,231 (published Apr. 20, 1988) and 264, 232(published Apr. 20, 1988) and in Japanese Kokai No. 62-87562 (publishedApr. 22, 1987) which disclose 2-azetidinone derivatives for bloodplatelet aggregation inhibitors.

This invention relates to novel 3-guanidinoalkyl-2-azetidinones aspotent thrombin and trypsin inhibitors. None of the references disclosesthe instant 3-guanidinoalkyl-2-azetidinones as serine proteaseinhibitors.

SUMMARY OF THE INVENTION

The present invention relates to new 3-guanidinoalkyl-2-azetidinones ofthe formula ##STR4## wherein: U and W can be the same or different andare selected from the group consisting of hydrogen and an aminoprotecting group;

n is 1 to 3;

X is a member selected from the group consisting of hydrogen,trialkylsilyl, arylsulfonyl, amino substituted arylsulfonyl,alkylsulfonyl, arylaminocarbonyl, alkylcarbonyl and arylcarbonyl; and

Y is a member selected from the group consisting of hydrogen,arylalkenyl, arylalkyl, formyl, carboxy, alkoxycarbonyl, acyloxy,arylthio, arylsulfinyl, arylsulfonyl, alkylthio, alkylsulfinyl,alkylsulfonyl, arylaminocarbonyl, the radical ##STR5## in which R ishydrogen, alkyl or arylalkyl, and the radical ##STR6## in which m is 1to 3 and R' is hydrogen or --CO₂ R" wherein R" is hydrogen, alkyl orarylalkyl.

The compounds are useful for anti-thrombin and anti-trypsin activitiesor as intermediates in the preparation of compounds having suchutilities.

DETAILED DESCRIPTION OF THE INVENTION

This application relates to novel azetidinone derivatives which arepotent inhibitors against serine proteases, in particular againstthrombin and trypsin. More specifically, it relates to3-guanidinoalkyl-2-azetidinones of the formula ##STR7## wherein: n is 1to 3;

X is a member selected from the group consisting of arylsulfonyl, aminosubstituted arylsulfonyl, alkylsulfonyl, arylaminocarbonyl,alkylcarbonyl and arylcarbonyl; and

Y is a member selected from the group consisting of hydrogen, arylalkyl,carboxy, alkoxycarbonyl, acyloxy, arylsulfonyl, alkylthio,alkylsulfonyl, arylaminocarbonyl, the radical ##STR8## in which R ishydrogen, alkyl or arylalkyl, and the radical ##STR9## in which m is 1to 3 and R' is hydrogen or --CO₂ R" wherein R" is hydrogen, alkyl, orarylalkyl. In prefered Formula A compounds of the present invention, nhas a value of 1 or 2, m is 2 or 3, R is hydrogen, and R' is carboxy orhydrogen.

In another aspect, this invention provides a method to control bloodcoagulation or to treat pancreatitis in an animal host which comprisesadministering to said host in need of such treatment a therapeuticallyeffective amount of a compound of Formula A.

Also included within the scope of the invention are nontoxicpharmaceutically acceptable salts, physiologically hydrolyzable esters,or solvates of the compounds of Formula A. The nontoxic pharmaceuticallyacceptable salts of the compounds of Formula A include salts withmineral acids such as hydrochloric, hydrobromic, phosphoric andsulfuric, or with organic carboxylic acids or sulfonic acids such asacetic, trifluoroacetic, citric, maleic, oxalic, succinic, benzoic,tartaric, fumaric, mandelic, ascorbic, malic, methanesulfonic,p-touenesulfonic, and the like. Preparation of these acid additionssalts is carried out by conventional techniques.

The invention also includes within its scope pharmaceutical compositionscontaining an effective thrombin or trypsin inhibiting amount of acompound of Formula A in combination with an inert pharmaceuticallyacceptable carrier or diluent. Such compositions may also contain otheractive agents and may be made up in any pharmaceutical form appropriatefor the desired route of administration. Examples of such compositionsinclude solid compositions for oral administration such as tablets,capsules, pills, powders and granules, liquid compositions such assolutions, suspensions, syrups or elixirs and preparations forparenteral administration such as sterile aqueous or non-aqueoussolutions, suspensions or emulsions. They may also be manufactured inthe form of sterile solid compositions which can be dissolved in sterilewater, physiological saline or some other sterile injectable mediumimmediately before use.

Optimal dosages and regimens of the compound of Formula A, wherein n, Xand Y are as previously defined, for a given mammalian host can bereadily ascertained by those skilled in the art. It will, of course, beappreciated that the actual dose used will vary according to theparticular composition formulated, the mode of application and theparticular situs, host and condition being treated. Many factors thatmodify the action of the drug will be taken into account including age,weight, sex, diet, time of administration, route of administration,condition of the patient, drug combinations, reaction sensitivities andseverity of the condition being treated. Administration can be carriedout continuously or periodically within the maximum tolerated dose.Optimal application rates for a given set of conditions can beascertained by those skilled in the art using conventional dosagedetermination tests in view of the above guidelines.

In another aspect, this application relates to a process for thepreparation of the compounds of Formula A. Compounds of this inventioncan be prepared from common intermediates 3a' and 3b' ##STR10## whereinp is 1 or 2; n is 1 to 3; R¹ is phenyl or alkyl substituted phenyl; Qand T can be the same or different amino protecting groups. For a rangeof amino protecting groups that can be used, see T. W. Greene,"Protective Groups in Organic Synthesis", 1981, John Wiley, New York,the disclosure of which is incorporated herein by reference. In thepresent invention, preferred protecting groups are phenylmethoxycarbonyl(Cbz) and t-butyloxycarbonyl groups. Compounds 3a' and 3b' can beprepared by a process which comprises the following steps or appropriatemodifications thereof:

(a) reacting an isourea of Formula i with an amino acid of Formula ii toafford an acid of Formula iii ##STR11## wherein R² refers to an alkylgroup, preferably a primary alkyl group, and n, Q and T are aspreviously defined;

(b) esterifying a compound of Formula iii to an ester of Formula iv##STR12## by activating the acid group in Formula iii with an activatingagent such as carbonyldiimidazole and reacting the resultant imidazolidewith an alcohol, preferably methanol, wherein R² refers to an alkylgroup, preferably methyl group;

(c) forming a trianion of a compound of Formula iv by using a strongbase such as lithium diisopropylamide, sodium hexamethyldisilizide,potassium hexamethyldisilizide, lithium hexamethyldisilizide, and thelike, and reacting said trianion with a silylimine of Formula v toafford an azetidinone of Formula vi, ##STR13## wherein R³, R⁴, and R⁵can independently be alkyl, phenyl, or alkyl substituted phenyl;

(d) hydrolyzing the nitrogen-silicon bond in a compound of Formula viunder acidic condition to afford a pair of stereoisomers of Formulas 3a'and 3b' in varying proportions which may be separated from each otherand isolated by separation techniques well known in the art, such as bysilica gel column chromatography.

As used herein, "alkyl" means a group containing from 1 to 4 straight orbranched carbon atoms; "alkoxy" means an alkyl group attached to oxygen;"acyloxy" refers to an alkyl group attached to carbonyl; "aryl" means aphenyl or naphthyl group which may be unsubstituted or substituted withone or more groups such as amino, nitro, or alkyl; "amino" refers to anamino group unsubstituted or subtituted with one or two alkyl radicals;and "alkenyl" refers to a group containing 2 to 4 carbon atoms with 1 or2 double bonds. Moreover, the invention embraces both E and Z formswhich arise from double bond(s) in alkenyl groups.

It will be appreciated that one or both carbon atoms at positions 3 and4 on the azetidinone core structure are chiral carbons, thus rendering acompound containing them to exist as optical isomers. It should beemphasized that the structures, as drawn in the specification, are notto be understood as representing specific enantiomers or tautomers (saidtautomers arising from different positions of hydrogen atoms or a doublebond in a guanidino moiety). The present invention intends to embraceall enantiomeric and tautomeric variations which are possible from thedrawn structures. Further, depending upon the structural orientation ofsubstituents attached to the 3 and 4 positions of the azetidinone, acompound may exist as either a cis or trans isomer. The presentinvention also intends to embrace both cis and trans isomers, unlessotherwise specified.

DESCRIPTION OF SPECIFIC EMBODIMENTS

In compounds of the present invention having the general formula,##STR14## specific examples of the group X include:

hydrogen,

t-butyldimethylsilyl,

p-toluenesulfonyl,

acetyl,

(5-dimethylamino)-1-naphthalenesulfonyl,

methylsulfonyl,

phenylaminocarbonyl,

t-butylcarbonyl, and

phenylcarbonyl.

Specific examples of the group Y include:

hydrogen,

2-phenylethenyl,

2-phenylethyl,

formyl,

carboxy,

methoxycarbonyl,

acetyloxy,

phenylthio,

phenylsulfonyl,

ethylthio,

ethylsulfonyl,

1-piperidinocarbonyl,

4-methylbenzeneaminocarbonyl,

[2-[(phenylmethoxy)carbonyl]-1-pyrrolidinyl]carbonyl,

[[2-oxo-2-(phenylmethoxy)ethyl]amino]carbonyl,

(2-carboxy-1-pyrrolidinyl)carbonyl, and

[(2-hydroxy-2-oxoethyl)amino]carbonyl.

The reaction schemes and specific examples which follow illustrate thesynthesis of representative compounds of the instant invention, and arenot to be construed as limiting the invention in sphere or scope. Themethods disclosed may be adopted to variations in order to producecompounds embraced by this invention but not specifically disclosed.Further, variations of the methods to produce the same compounds insomewhat different fashion will also be evident to one skilled in theart.

Referring to Scheme 1, the intermediate 1 is prepared by reactingN,N'-dicarbobenzyloxy-5-methylisothiourea with 5-aminovaleric acid.Compound 1 is esterified to form the corresponding methyl ester 2 whichis cyclized by reaction with N-(trimethylsilyl)cinnamilyldenimine toform a mixture of isomers 3a and 3b. Isomers 3a and 3b are separated andthen are individually converted to compounds 4a and 4b by reaction witht-butyldimethylsilyl chloride. Compounds 4a and 4b are individuallyozonized to form aldehydes 5a and 5b which are treated with Jonesreagent to form compounds 6a and 6b. ##STR15##

Referring to Scheme 2, compound 6a is esterified to give compound 7.Compound 7 is treated with various reagents as specified hereinafter toreplace the proton at the 1-position to give compounds 8, 9, 10, 11 and12, each of which is then catalytically hydgrogenolyzed to remove theamino protecting groups, to yield compounds I, II, III, IV and V,respectively. ##STR16##

Referring to Scheme 3, compound 6a is reacted with lead tetraacetate toplace an acetyloxy group on the 4-position to form compound 13. Compound13 is reacted with sodium thiophenoxide to produce compound 14, which isreacted with acetyl chloride to yield compound 15. Compound 15 isoxidized to give compound 16, which is then catalytically hydrogenolyzedto remove the amino protecting groups to yield compound VI.Alternatively, compound 13 is reacted with sodium salt of ethanethiol toproduce compound 17, which is then reacted with acetyl chloride to yieldcompound 18. Removal of the amino protecting groups by catalytichydrogenolysis from compound 18 yields compound VII. ##STR17##

Referring to Scheme 4, compound 17 is treated with Raney-Nickel to formcompound 19. Compound 19 is treated with various reagents as describedhereinafter to replace the proton at the 1-position to give compounds 20and 21, each of which is then catalytically hydrogenolyzed to remove theamino protecting groups to yield compounds VIII and IX, respectively.##STR18##

Referring to Scheme 5, compound 18 is oxidized to give compound 22 whichis then catalytically hydrogenolyzed to remove the amino protectinggroups to yield compound X. ##STR19##

Referring to Scheme 6, compound 6a is reacted with piperidine to givecompound 23. Compound 23 is treated with various reagents as specifiedhereinafter to replace the proton at the 1-position to give compounds24, 25 and 26, each of which is then catalytically hydrogenolyzed withhydrogen to remove the amino protecting groups to yield compounds XI,XII and XIII, respectively. ##STR20##

Referring to Scheme 7, compound 3a is reacted with acetyl chloride togive compound 27, which is ozonized to form aldehyde 28. Aldehyde 28 istreated with Jones reagent to form compound 29 from which the aminoprotecting groups are removed by catalytic hydrogenolysis to yieldcompound XIV. Compound 27 is also hydrogenated to yield compound XV.Compound 29 is also reacted with p-toluidene to form compound 30, whichis catalytically hydrogenolyzed to remove the amino protecting groups toyield compound XVI. ##STR21##

Referring to Scheme 8, the intermediate 31 is prepared by reactingN,N'-dicarbobenzyloxy-S-methylisothiourea with 6-aminocaproic acid.Compound 31 is esterified to form the corresponding methyl ester 32which is cyclized by reaction with N-(trimethylsilyl)benzaldimine toform compound 33. Compound 33 is converted to compound 34 by reactionwith t-butyldimethylsilyl chloride. Compound 34 is ozonized to formaldehyde 35 which is treated with Jones reagent and subsequentlyesterified to form compound 36. Compound 36 is reacted with dansylchloride to give compound 37 from which the amino protecting groups areremoved by catalytic hydrogenolysis to yield compound XVII. ##STR22##

Referring to Scheme 9, compound 29 is converted to compounds 38 and 39,which are catalytically hydrogenolyzed to yield compounds XVIII and XIX,respectively. In the scheme, the symbol "s" designates the configurationat the carbon atom according to the well-known Cahn-Ingold-Trelogsystem. ##STR23##

Referring to Scheme 10, compound 13 is treated with various reagents asdescribed hereinbelow to replace the proton at the 1-position to givecompounds 40, 41 and 42, each of which is then catalyticallyhydrogenolyzed to remove the amino protecting groups to yield compoundsXX, XXI and XXII, respectively. ##STR24##

Referring to Scheme 11, compound 6b is reacted with piperidine to givecompound 43 which is reacted with acetyl chloride to give compound 44.Removal of the amino protecting groups by catalytic hydrogenolysis fromcompound 44 yields compound XXIII. ##STR25##

EXPERIMENTALS

In the examples which follow, melting points were recorded on aThomas-Hoover capillary melting point apparatus and are uncorrected. Allspectra were determined in CDCl₃, DMSO-d₆, CD₃ OD, or D₂ O unlessotherwise indicated and chemical shifts are reported in ppm unitrelative to the deuterated solvents used. Interproton coupling constantsare reported in Hertz (Hz). Splitting patterns are designated asfollows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;br, broad peak; dd, doublet of doublet; and dq, doublet of quartet.Infrared (IR) spectra are reported in reciprocal centimeters (cm⁻¹).Relative intensities are indicated as follows: s (strong), m (medium), w(weak), and br (broad).

Analytical thin-layer chromatography (TLC) was carried out on precoatedsilica gel plates (60F-254) and visualized using UV light, iodinevapors, and/or staining with methanolic or ethanolic phosphomolybdicacid (2% solution). Silica gel chromatography was performed in a glasscolumn using finely divided silica gel (32-63 μm on silica gel-H) and ata pressure somewhat above atmospheric pressure with the indicatedsolvents. Evaporation and/or concentration of solvents were performedunder a reduced pressure. EtOAC is ethyl acetate; Et₂ O or ether isdiethyl ether; AcOH is acetic acid; THF is tetrahydrofuran; DMF isdimethylformamide; MCPBA is m-chloroperbenzoic acid; U is NIH units; andCbz is phenylmethoxylcarbonyl (or otherwise known as carbobenzyloxy)group. Celite is a registered trademark of the Johns-Manville ProductsCorporation for diatomaceous earth.

All hydrogenolysis was conducted under one atmospheric pressure ofhydrogen unless otherwise specified.

EXAMPLE 1 5-[N',N"-Di(Cbz)guanidino]pentanoic acid (1)

To a methanolic solution (1,000 mL) of N,N'-dicarbobenzyloxy-5-methyl-isothiourea [323 g, 0.9 mol, for thesynthesis of N, N'-dicarbobezyloxy-5-methyl-isothiourea see K. Nowah etal., Rocz. Chem., 43, 1953-1960 (1969)] and triethylamine (75.1 g, 0.74mol) was added 5-aminovaleric acid (86.9 g, 0.74 mol) portionwise. Theresultant suspension was refluxed for 1.5 h and concentrated. Theresidue was partitioned between EtOAc and saturated aqueous NaHCO₃solution. The aqueous layer was acidified to pH 2.0 with hydrochloricacid and extracted with EtOAc. The organic layer was dried (MgSO₄) andconcentrated. The residual solid was recrystallized (EtOAc/hexane) toafford 135.1 g (54% based on 5-aminovaleric acid), mp 102°-103° C.

¹ H NMR(CDCl₃) δ: 8.35 (1H, br s), 7.43-7.25 (10H, m), 5.18 (2H, s),5.13 (2H, s), 3.45 (2H, br q, J=6.5), 2.4 (2H, br t, J=4.8), 1.80 (4H,m);

IR (KBr): 1735, 1720 cm⁻¹ ;

Anal. Calcd. for C₂₂ H₂₅ N₃ O₆ : C, 61.81; H, 5.89; N, 9.83. Found: C,61,39; H, 6.29; N, 9.92.

EXAMPLE 2 5-[N',N"-di(Cbz)guanidino]pentanoic acid methyl ester (2)

To a 0° C. THF (700 mL) solution of compound 1 (133 g, 0.3 mol) wasadded carbonyldiimidazole (55.3 g, 0.34 mol). After the addition wascomplete, the resultant solution was allowed to warm to room temperatureand stirred for 45 min. Anhydrous methanol (100 mL) was added. Thesolution was stirred for 16 h and concentrated. The residue wastriturated with ether (400 mL). The supernatant was separated. Theprocess was repeated with two more portions of ether (2×200 mL). Theether phases were combined, washed with H₂ O, dried (MgSO₄), andconcentrated. The residue was purified by silica gel chromatography (Et₂O:hexane/1:1) to afford 130 g (95%) of the title product, mp 36°-39° C.

¹ H NMR (CDCl₃) δ: 8.36-8.28 (1H, br s), 7.46-7.21 (10H, m), 5.15 (2H,s), 5.09 (2H, s), 3.64 (3H, s), 3.42 (2H, br q, J=4.9), 2.32 (2H, br t,J=4.9), 1.24-1.50 (4H, m);

IR (KBr): 1740 cm⁻¹ ;

Anal. Calcd. for C₂₃ H₂₇ H₂₇ N₃ O₆ : C, 62.54; H, 6.15; N, 9.51. Found:C, 62.64 H, 6.03; N, 9.37.

EXAMPLE 3trans-4-(2-Phenylethenyl)-3-[3-[N',N"-di(Cbz)guanidino]propyl]-2-azetidinone(3a) andcis-4-(2-phenylethenyl)-3-[3-[N',N"-di(Cbz)guanidino]propyl]-2-azetidinone(3b)

To a -40° C. THF (800 mL) solution of 1,1,1,3,3,3-hexamethyldisilazane(110 g, 0.69 mol) was added 2.5M hexane solution of n-butyllithium (272mL, 0.69 mol). The resultant solution was cooled to -78° C. followed bythe addition of THF (300 mL) solution of compound 2 (106 g, 0.24 mol).After the addition was complete, the reaction mixture was allowed towarm to -30° C. and kept at this temperature for 1 h followed by coolingto -78° C. THF (100 mL) solution of N-(trimethylsilyl)cinnamilydenimine[51.5 g, 0.25 mol, for the synthesis ofN-(trimethylsilyl)cinnamilydenimine, see example 67] was added dropwise,and the resulting solution was kept at -20° C. for 16 h. (For a relatedaddition of the β-hydroxy ester danion to cinnamilydenimine, see G.Cainelli et al., Tetrahedron Lett., 26, 937 (1985)). The light greenreaction mixture was concentrated to half of its original volume anddiluted with ether. The solution was stirred with 5N HCl; enough acidwas used to maintain the pH of the aqueous phase at 2.0. The organicphase was separated, washed with H₂ O and brine, dried (MgSO₄), andconcentrated- The residue was purified by silica gel chromatography toafford 50 g (38%) of the trans isomer, 5.7 g (4.4%) of the cis isomerand 26.5 g (20%) of the mixture of cis and trans isomers.

trans isomer 3a: mp 101° C.;

¹ H NMR (CDCl₃) δ: 8.43 (1H, br t, J=4.0), 7.50-7.24 (15H, m), 6.66 (1H,d, J=14.5), 6.58 (1H, s), 6.27 (1H, dd, J=14.5, 8.0), 5.22 (2H, s), 5.15(2H, s), 3.97 (1H, br d, J=8.0), 3.56-3.45 (2H, m), 3.01 (1H, m),1.85-1.70 (4H, m);

IR (film): 1734 cm⁻¹ ;

Anal. Calcd. for C₃₁ H₃₂ N₄ O₅ : C, 68.86; H, 5.96; N, 10.36. Found: C,68.91; H, 5.94; N, 10.29.

cis isomer 3b:

¹ H NMR (CDCl₃) δ: 8.27 (1H, br t, J=5.6), 7.37-7.14 (15H, m), 6.12 (1H,d, J=14.5), 6.12 (1H, dd, J=14.5, 7.2), 5.95 (1H, br s), 5.07 (2H, s),5.05 (2H, s), 4.32 (1H, br t, J=7.2), 3.48-3.25 (3H, m), 1.78-1.50 (4H,m);

IR (film): 1760, 1745 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₁ H₃₃ N₄ O₅ : (M+H) 541.2451. Found:541.2441.

EXAMPLE 4trans-4-(2-Phenylethenyl)-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-t-butyldimethylsilyl-2-azetidinone(4a)

To a DMF (40 mL) solution of compound 3a (7.8 g, 14.4 mmol) andtriethylamine (2.1 g, 21 mmol) was added t-butyldimethylsilyl chloride(3.12 g, 21 mmol). The reaction mixture was stirred at room temperaturefor 15 h and partitioned between Et₂ O and H₂ O. The organic layer wasseparated, washed with additional H₂ O, dried (MgSO₄), and concentrated.The residue was purified by silica gel chromatography (Et₂ O:hexane/1:1)to afford 8.3 g (88% of the title product.

¹ H NMR (CDCl₃) δ: 8.37 (1H, t, J=4.5) 7.45-7.23 (15H, m), 6.58 (1H, d,J=16.0), 6.17 (1H, dd, J=16.0, 9.9), 5.6 (2H, s), 5.13 (2H, s), 3.86(1H, dd, J=9.9, 1.5), 3.45-3.41 (2H, m), 3.04-2.95 (1H, m), 1.90-1.65(4H, m), 0.88 (9H, s), 0.16 (3H, s), 0.1 (3H, s);

Anal. Calcd. for C₃₇ H₄₆ N₄ O₅ Si: C. 67.86, H, 7.08; N, 8.56. Found: C,67.12; H, 7.06; N, 8.21;

High resolution FAB MS Calcd. for C₃₇ H₄₇ N₄ O₅ Si: (M+H) 655.3316.Found: 655.1295.

EXAMPLE 5 Step Atrans-4-Formyl-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-t-butyldimethylsilyl-2-azetidinone(5a)

Ozone was passed through a -78° C. CH₂ Cl₂ (650 mL) solution of compound4a (55 g, 84 mmol) until a blue color was maintained. Excess ozone waspurged with N₂. Dimethyl sulfide (60 mL) was added. The solution wasallowed to warm to room temperature and to stand for 48 h. The solutionwas concentrated to afford the title compound which was not furtherpurified but used directly in step B.

¹ H NMR (CDCl₃) δ: 9.63 (1H, d, J=5.3), 8.40 (1H, br t, J=5.3),7.60-7.25 (10H, m), 5.17 (2H, s), 5.13 (2H, s), 3.67 (1H, m), 3.53-3.40(2H, m), 3.28-3.21 (1H, m), 1.93-1.65 (4H, m), 0.97 (9H, s), 0.2 (3H,s), ) 0.13 (3H, s).

Step Btrans-4-Carboxy-3-[3-[N',N"-di(Cbz)guanidino]propyl]-2-azetidinone (6a)

The aldehyde 5a prepared in Step A was dissolved in acetone (75 mL).Freshly prepared Jones reagent was added dropwise to the acetonesolution, maintaining the temperature between 20°-25° C., until the TLCindicated the disappearance of the starting aldehyde. The resultantsuspension was concentrated to half of its original volume andpartitioned between EtOAc and H₂ O. The organic phase was dried (Na₂SO₄) and concentrated. The residual solid was recrystallized fromEtOAc/hexane to afford 8.8 g (54% from 4a) of the title compound as agreenish solid, mp 172°-173° C.

¹ H NMR (CDCl₃) δ: 8.43(1H, br s), 8.24 (1H, s), 7.45-7.20 (10H, m),5.17 (2H, s), 4.99 (2H, s), 3.74 (1H, d, J=1.8), 1.70-1.50 (4H, m);

High resolution FAB MS Calcd. for C₂₄ H₂₇ N₄ O₇ : (M+H) 483.1890. Found:683.1880.

EXAMPLE 6cis-4-(2-Phenylethenyl)-3-[3-[N',N"-di(Cbz)guanidino]-propyl]-1-t-butyldimethylsilyl-2-azetidinone(4b)

To a DMF (30 mL) solution of compound 3b (5.6 g, 10.4 mmol) was addedtriethylamine (1.73 g 17 mmol) and t-butyldimethylsilyl chloride (1.86g, 12 mmol). The resultant reaction mixture was stirred for 1 h followedby partitioning between Et₂ O and H₂ O. The organic layer was dried(MgSO₄) and concentrated. The residue was purified by silica gelchromatography (Et₂ O:hexane/3:2) to afford 4.73 g (78.6%) of the titleproduct.

¹ NMR (CDCl₃) δ: 8.30 (1H, br t, J=5.4), 7.39-7.21 (15H, m), 6.57 (1H,d, J=15.2), 6.02 (1H, dd, J=15.2, 8.4), 4.11 (2H, s), 5.08 (2H, s), 4.22(1H, dd, J=8.4, 6.09), 3.50-3.26 (3H, m), 1.84-1.51 (4H, m), 0.94 (9H,s), 0.23 (3H, s), 0.14 (3H, s);

High resolution FAB MS Calcd. for C₃₇ H₄₇ N₄ O₅ Si: (M+H) 655.3316.Found: 655.3323.

EXAMPLE 7 Step Acis-4-Formyl-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-t-butyldimethysilyl-2-azetidinone(5b)

Ozone was passed through a -78° C. methylene chloride (100 mL) solutionof compound 4b (4.73 g, 7.22 mmol) until a blue color was maintained.Excess ozone was purged with nitrogen. Dimethyl sulfide (10 mL) wasadded. The solution was allowed to stand for 16 h and concentrated toafford the title product which was not further purified but useddirectly in Step B.

Step B cis4Carboxyl-3-[3-[N',N"-di(Cbz)guanidino]propyl]-2-azetidinone(6b)

Compound 5b prepared in Step A was taken up in acetone (50 mL). Jonesreagent was added dropwise until the orange color persisted. Theresultant reaction mixture was stirred for an additional 15 min andconcentrated. The residue was partitioned between H₂ O and EtOAc. Theorganic layer was dried (MgSO₄) and concentrated. The residue waspurified by silica gel chromatography (EtOAc:Et₂ O:AcOH/1:4:0.05) toafford 1.8 g (51%) of the title product.

¹ H NMR (CDCl₃) δ: 8.4 (1H, br s), 7.45-7.10 (10H, m), 6.70 (1H, br s),5.17 (2H, s), 5.07 (2H, s), 4.19 (1H, d, J=6.9), 3.50-3.10 (3H, m),1.80-1.50 (4H, m);

High resolution FAB MS Calcd. for C₂₄ H₂₇ N₄ O₇ : (M+H) 483.1880. Found:483.1878.

EXAMPLE 8trans-4-Methoxycarbonyl-3-[3-[N',N"-di(Cbz)guanidino]-propyl]-2-azetidinone(7)

An ether solution of diazomethane was added to a methylene chloride (50mL) solution of compound 6a (4.69, 9.72 mmol) until the yellow color wasmaintained. Excess diazomethane was destroyed by letting it react withacetic acid. The solution was concentrated and the residue was purifiedby silica gel chromatography (EtOAc:Et₂ O/9:1) to afford 3.41 g (71%) ofthe title product.

¹ H NMR (CDCl₃) δ: 8.38 (1H, br t, J=5.3), 7.45-7.25 (10H, m), 6.27 (1H,br s), 5.18 (2H, s), 5.13 (2H, s), 3.88 (1H, d, J=2.4), 3.77 (3H, s),3.54-3.42 (2H, m), 3.31-3.22 (1H, m), 1.95-1.70 (4H, m);

Anal. Calcd. for C₂₅ H₂₈ N₄ O₇ : C, 60.47; H, 5.68; N, 11.28. Found: C,60.36; H, 5.76; N, 11.26.

EXAMPLE 9trans-4-Methoxycarbonyl-3-[3-[N',N"-di(Cbz)guanidino]-propyl]-1-p-toluenesulfonyl-2-azetidinon e (8)

To a -78° C. THF (5 mL) solution of compound 7 (4.36 mg, 0.67 mmol) wasadded 1N THF solution of sodium bis(trimethylsilyl)amide (0.67 mL, 0.67mmol), and the resultant solution was stirred for 15 min.p-Toluenesulfonyl chloride (134 mg, 0.70 mmol) was added, and themixture was stirred at -78° C. for 4 h and at -20° C. for 21/2 days. Thereaction mixture was partitioned between EtOAc and H₂ O. The organiclayer was dried (Na₂ SO₄) and concentrated. The residue was purified bysilica gel chromatography (Et₂ O:hexane/3:2) to afford 210 mg (48%) ofthe title product.

¹ H NMR (CDCl₃) δ: 8.32 (1H, br t, J=6.1 ), 7.88 (1H, d, J=8.4),7.40-7.20 (14H, m), 5.10 (2H, s), 5.06 (2H, s), 4.98 (1H, br s), 4.28(1H, d, J=1.5), 3.72 (3H, s), 3.45-3.33 (2H, m), 3.23-3.13 (1H, m), 2.42(3H, s), 1.85-1.55 (4H, m);

IR (film): 1805, 1735 cm⁻¹ ;

Anal. Calcd. for C₃₂ H₃₄ N₄ O₉ S: C, 58.77; H, 5.14; N, 8.14 Found: C,59.06; H, 5.26; N, 8.61.

EXAMPLE 10trans-4-Methoxycarbonyl-3-(3-guanidinopropyl)-1-p-toluenesulfonyl-2-azetidinonehydrochloride salt (I)

An ethyl acetate/methanol (2.5 mL/2.5 mL) solution of compound 8 (197mg, 0.3 mmol) and 1N HCl (0.6 mL), containing palladium on carboncatalyst, was stirred under one atmosphere of hydrogen until TLCindicated the disappearance of the starting 8. The suspension wasfiltered through a pad of Celite, and the filtrate was concentrated toafford 30 mg (24%) of the title product as a foam.

¹ H NMR (CD₃ OD) δ: 7.88 (2H, d, J=8.5 ), 7.44 (2H, d, J=8.5), 4.47 (1H,d, J=3.6), 3.74 (3H, s), 3.42 (1H, dt, J=7.2, 3,6), 3.16 (2H, t, J=6.6),2.46 (3H, s), 1.86-1.56 (4H, m);

IR (film): 1800, 1755 cm⁻¹ ;

Anal. Calcd. for C₁₆ H₂₄ N₄ O₅ Cl: C, 44.18; H, 5.33; N, 12.88; Cl,8.15; Found: C, 43.79; H, 5.62; N, 12.52; Cl, 8.01;

High resolution FAB MS Calcd. for C₁₆ H₂₄ N₄ O₅ : (M+H) 383.1389. Found:383.1376.

EXAMPLE 11trans-4-Methoxycarbonyl-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-acetyl-2-azetidinone(9)

Acetyl chloride (9.5 g, 0.12 mol) was added to a -10° C. methylenechloride (50 mL) solution of compound 7 (2.0 g, 4.0 mmol). Triethylamine(12.3 g, 0.12 mol) was added dropwise with vigorous stirring of thereaction mixture. After the addition was complete, the resultantsuspension was stirred for an additional 2 h at room temperature andthen washed with H₂ O. The organic phase was separated, washed withsaturated aqueous NaHCO₃ solution, dried (Na₂ SO₄), and concentrated.The residue was purified by silica gel chromatography (CH₂ Cl₂:MeOH/99.5:0.5) to afford 700 mg (36%) of the title product.

¹ H NMR δ: 8.32 (1H, br t, J=5.0), 7.37-7.20 (10H, m), 5.16 (2H, s),5.07 (2H, s), 4.08 (1H, d, J=2.3), 3.37 (3H, s), 3.47-3.38 (2H, m),3.26-3.15 (1H, m), 2.34 (3H, s), 1.93-1.60 (4H, m);

IR (film): 1800, 1740, 1720 cm⁻¹ ;

High resolution FAB MS Calcd. for C₂₇ H₃₁ N₄ O₈ : (M+H) 539.2125. Found:539.2162.

EXAMPLE 12 trans-4-Methoxycarbonyl-3-(3-guanidinopropyl)-1-acetyl-2-azetidinone hydrochloride salt (II)

A methanol/ethyl acetate (2.5 mL/2.5mL) solution of compound 9 (313 mg,0.58 mmol) and 1N HCl (0.58 mL) was stirred under a hydrogen atmospherewith 10% palladium on carbon catalyst. When the starting material haddisappeared as determined by TLC, the reaction mixture was passedthrough a pad of Celite and the filtrate was concentrated to afford 150mg (84%) of the title product as a yellow foam.

¹ H NMR (CD₃ OD) δ: 4.27 (1H, d, J=3.1) 3.78 (3H, s), 3.50-3.10 (3H, m),2.35 (3H, s), 1.90-1.60 (4H, m);

IR (film): 1800 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₁ H₁₈ O₄ : (M+H) 271.1406. Found:271.1414.

EXAMPLE 13trans-4-Methoxycarbonyl-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-[(5-dimethylamino)-1-naphthalenesulfonyl]-2-azetidinone(10)

To a -78° C. THF (5 mL) solution of compound 7 (1.03 g, 2.08 mmol) wasadded 1N THF solution of sodium bis(trimethylsilyl)amide (2.08 mL, 2.08mmol), and the resultant solution was stirred for 20 min. Dansylchloride (0.5 g, 2.08 mmol) was added. The mixture was allowed to warmto room temperature, stirred for 1 h, and partitioned between EtOAc andpH 7.0 aqueous buffer. The organic layer was dried (Na₂ SO₄) andconcentrated. The residue was purified by silica gel chromatography (Et₂O) to afford 0.45 g (30%) of the title product.

¹ H NMR (CDCl₃) δ: 8.61 (1H, d, J=8.4), 8.47 (1H, d, J=9.2), 8.28 (1H,d, J=6.9), 7.63-7.49 (2H, m), 7.45-7.15 (11H, m), 5.16 (2H, s), 5.08(2H, s), 4.22 (1H, d, J=2.3), 3.46 (3H, s), 3.42-3.33 (2H, m), 3.31-3.24(1H, m), 2.84 (6H, s), 1.86-1.53 (4H, m);

IR (KBr): 1810, 1755 cm⁻¹ ;

FAB MS: 730 (M+H, base ).

EXAMPLE 14trans-4-Methoxycarbonyl-3-(3-guanidinopropyl)-1-[(5-dimethylamino)-1-naphthalenesulfonyl]-2-azetidinonehydrochloride salt (III)

A methanol/ethyl acetate (5 mL/2 mL) solution of compound 10 (0.45 g,0.62 mmol) and 1N HCl (0.7 mL), containing 10% palladium on carboncatalyst, was stirred under hydrogen. When the starting material haddisappeared as determined by TLC (25 min), the reaction mixture waspassed through a pad of Celite and the filtrate was concentrated toafford 276 mg (89%) of the title product.

¹ H NMR (CD₃ OD) δ: 8.46 (1H, d, J=8.4), 8.38 (1H, d, J=9.2), 8.08 (1H,d, J=7.7), 7.53-7.40 (2H, m), 7.28 (1H, br d, J=6.9), 4.20 (1H, d,J=2.3), 3.19 (3H, s), 3.09-1.84 (3H, m), 2.77 (6H, s), 1.60-1.30 (4H,m);

IR (KBr): 1805, 1755 cm⁻¹ ;

High resolution FAB MS Calcd. for C₂₁ H₂₈ N₅ O₅ S: (M+H) 462.1811.Found: 462.1801.

EXAMPLE 15trans-4-Methoxycarbonyl-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-methylsulfonyl-2-azetidinone(11)

To a vigorously stirred methylene chloride (10 mL) solution of compound7 (0.8 g, 1.6 mmol) and triethylamine (4.86 g, 48 mmol ) was addedmethanesulfonyl chloride (5.52 g, 48 mmol) dropwise. The reactionmixture was diluted with additional CH₂ Cl₂ and washed with pH 7.0aqueous buffer. The organic phase was dried (Na₂ SO₄) and concentrated.The residue was purified by silica gel chromatography (CH₂ Cl₂ :MeOH/99:1) to afford 340 mg (37%) of the title compound.

¹ H NMR (CDCl₃) δ: 7.40-7.13 (10H, m), 5.16 (2H, s), 5.10 (2H, s), 4.35(1H, d, J=3.18), 3.80 (3H, s), 3.46 (2H, d, J=6.6), 3.3 (3H, s),3.28-3.25 (1H, m), 1.95-1.73 (4H, m);

IR (film): 1808, 1745 cm⁻¹ ;

High resolution FAB MS Calcd. for C₂₆ H₃₁ N₄ O₉ S: (M+H) 575.1812.Found: 575.1801.

EXAMPLE 16trans-4-Methoxycarbonyl-3-(guanidinopropyl)-1-methylsulfonyl-2-azetidinonehydrochloride salt (IV)

A methanol solution of compound 11 (0.29 g, 0.5 mmol) and 1N HCl (0.5mL), containing 10% palladium on carbon, was stirred under 35 psi ofhydrogen atmosphere for 2 h. The suspension was filtered through a padof Celite and the filtrate was concentrated to afford 52 mg (30%) of thetitle product.

¹ H NMR (CD₃ OD) δ: 4.56 (1H, s), 3.83 (3H, s), 3.54-3.18 (3H, m), 3.30(3H, s), 1.96-1.63 (4H, m);

IR (film): 1810, 1755 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₀ H₁₉ O₅ N₄ S: (M+H) 307.1076.Found: 307.1066.

EXAMPLE 17trans-4-Methoxycarbonyl-3-[3-[N',N"-di(Cbz)ganidino]propyl]-1-phenylaminocarbonyl-2-azetidinone(12)

To a THF (10 mL) solution of compound 7 (690 mg, 1.38 mmol) at -78° C.was added 1N THF solution of sodium bis(trimethylsilyl) amide (0.149 mL,0.149 mmol), and the resultant mixture was stirred for 10 min.Phenylisocyanate (0.149 mL, 1.38 mmol) was added, followed by warmingthe solution to room temperature and stirring for 1 h. The solution wasconcentrated, and the residue was partitioned between pH 4.0 aqueousbuffer and ethyl acetate. The organic phase was dried (MgSO₄) andconcentrated. The residue was purified by silica gel chromatography (Et₂O:hexane/3:1) to afford 450 mg (53%) of the title product as a whitefoam.

¹ H NMR (CDCl₃) δ: 7.53-7.03 (15H, m), 5.17 (2H, s), 5.05 (2H, s), 4.23(1H, d, J=2.3), 3.78 (3H, s), 3.53-3.40 (2H, m), 3.36-3.26 (1H, m),2.0-1.68 (4H, m);

IR (KBr): 1780, 1732 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₂ H₃₄ H₅ O₈ : (M+H) 616.2407. Found:616.2413.

EXAMPLE 18trans-4-Methoxycarbonyl-3-(3-guanidinopropyl)-1-phenylaminocarbonyl-2-azetidinonehydrochloride salt (V)

A methanol/ethyl acetate (2.5 mL/2.5 mL) solution of compound 12 (421mg, 0.68 mmol) and 1N HCl (0.68 mL), containing 10% palladium on carbon,was stirred under a hydrogen atmosphere for 1 h. The reaction mixturewas filtered through a pad of Celite, and the filtrate was concentratedto afford 270 mg (quantitative) of the title product as a yellow foam.

¹ H NMR (CD₃ OD) δ: 7.49-7.09 (5H, m), 4.38 (1H, d, J=2.8), 3.82 (3H,s), 3.51-3.21 (3H, m), 1.98-1.73 (4H, m);

IR (KBr): 1780, 1740 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₆ H₂₂ O₄ N_(s) : (M+H) 348.1672.Found: 348.1679.

EXAMPLE 194-Acetyloxy-3-[3-[N',N"-di(Cbz)guanidino]propyl]-2-azetidinone (13)

Compound 6a (26.1 g, 54 mmol) was dissolved in a solution of acetic acid(105 mL) and DMF (15 mL) with warming. The resultant greenish solutionwas cooled to room temperature, followed by addition of leadtetraacetate (71.5 g, 160 mmol) in small portions. The suspension wasstirred for 5 h at 60° C. and concentrated. The residue was resuspendedin methylene chloride and filtered through a pad of Celite. The filtratewas concentrated, and the residue was purified by silica gelchromatography (Et₂ O:hexane/3:2) to afford 15.5 g (57%) of the titleproduct as a mixture of trans and cis (2:1) isomers.

¹ H NMR (CDCl₃) δ: 7.37-7.18 (10H, m), 5.78(1/3H, d, J=3.0), 5.46 (2/3H, br s), 5.13 (2H, s), 5.07 (2H, s), 3.45-3.36 (2H, m), 3.15-3.08 (1H,m), 2.07 (1H, s), 2.05 (2H, s);

High resolution FAB MS Calcd. for C₂₅ H₂₉ N₄ O₇ : 497.2036. Found:497.2031.

EXAMPLE 20trans-4-Phenylthio-3-[3-[N',N"-di(Cbz)guanidino]propyl]-2-azetidinone(14)

To a THF (20 mL) suspension of sodium thiophenoxide formed from NaH (190mg, 4.90 mmol, 60% oil dispersion) and thiophenol (0.54 g, 4.9 mmol) wasadded compound 13 (1.22 g, 2.45 mmol). The resultant solution wasstirred for 30 min and concentrated under vacuo. The residue waspartitioned between ethyl acetate and water. The organic layer was dried(MgSO₄) and concentrated. The residue was purified by silica gelchromatography (Et₂ O:hexane/3:1) to afford 450 mg (34%) of the titleproduct.

¹ H NMR (CDCl₃) δ: 8.37-8.26 (1H, br s), 7.48-7.20 (15H, m), 6.47 (1H,br s), 5.17 (2H, s), 5.10 (2H, s), 4.64 (1H, br s), 3.52-3.36 (2H, m),3.07-2.96 (1H, m), 3.07-2.96 (1H, m), 1.85-1.55 (4H, m);

IR (film): 1770, 1740 cm⁻¹.

EXAMPLE 21 Step Atans-4-Phenylthio-3-[3-[N',N"-di(Cbz)guanidino]-propyl]-1-acetyl-2-azetidinone(15)

To a -78° C. THF (7 mL) solution of compound 14 (450 mg, 0.82 mmol) wasadded 1N THF solution of sodium bis(trimethylsilyl)amide (0.8 mL)dropwise. The resultant solution was stirred for 10 min, followed by theaddition of acetyl chloride (0.054 mL, 0.76 mmol). The resultantsolution was warmed to room temperature and stirred for an additional 30min and concentrated- The residue was partitioned between ethyl acetateand H₂ O. The organic phase was dried (MgSO₄) and concentrated to afford450 mg (93%, crude yield) of the title compound which was directly usedin Step B.

¹ H NMR (CDCl₃) δ: 8.27 (1H, br t, J=5.3), 7.53-7.20 (15H, m), 5.12(2H,s), 5.08 (2H, s), 4.82 (1H, d, J=2.3), 3.38 (2H, q, J=6.9), 3.03 (1H,dt, J=8.4, 2.3), 2.32 (3H, s), 1.80-1.52 (4H, m);

IR (film): 1800, 1730 (br) cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₁ H₃₃ N₄ O₆ S: 589.2121. Found:589.2111.

Step Btrans-4-Phenylsulfonyl-3-[3-[N',N"-di(Cbz)guanidino]-propyl]-1-acetyl-2-azetidinone(16)

To a methylene chloride (10 mL) solution of compound 15 (450 mg, 0.76mmol) was added MCPBA (323 mg, 1.52 mmol, 80% purity), and the resultantsolution was stirred for 20 min. The solution was concentrated, and theresidue was partitioned between saturated aqueous NaHCO₃ solution andethyl acetate. The organic layer was dried (MgSO₄) and concentrated. Theresidue was chromatographed by silica gel chromatography (Et₂O:hexane/3:1) to afford 210 mg (44%) of the title compound.

¹ H NMR (CDCl₃) δ: 8.40-8.30 (1H, br t), 7.92-7.20 (15H, m), 5.15 (2H,s), 5.08 (2H, s), 4.90 (1H, d, J=2.3), 3.89-3.80 (1H, m), 3.55-3.40 (2H,m), 2.28 (3H, s), 1.95-1.65 (4H, m);

IR (KBr): 1815, 1715 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₅ H₂₁ N₄ O₄ S: 353.128. Found:353.1281

EXAMPLE 22 trans-4-Phenylsulfonyl-3-guanidinopropyl-1-acetyl-2-azetidinehydrochloride salt (VI)

A methanol/ethyl acetate (12.5 mL/12.5 mL) solution of compound 16 (210mg, 0.34 mmol) and 1N HCl (0.34 mL), containing 10% palladium on carbon,was stirred under a hydrogen atmosphere. After the disappearance of thestarting material as determined by TLC, the reaction mixture wasfiltered through a pad of Celite and the filtrate was concentrated toafford 100 mg (76%) of the title product as colorless foam.

¹ H NMR (CD₃ OD) δ: 8.03-7.60 (5H, m), 5.29 (1H, d, J=2.9), 3.83-3.76(1H, m), 3.22 (2H, t, J=6.9), 2.26 (3H, s), 2.01-1.70 (4H, m);

IR (KBr): 1815, 1730 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₅ H₂₁ N₄ O₄ S: (M+H) 353.1284.Found: 353.1281.

EXAMPLE 23trans-4-Ethylthio-3-[3-[N',N"-di(Cbz)guanidino]propyl]-2-azetidinone(17)

To a 0° C. suspension of NaH (60% oil dispersion, 200 mg, 498 mmol) inTHF (20 ml) was added ethanethiol (0.37 mL, 4.98 mmol). The resultantmixture was stirred for 10 min, cooled to -20° C., and compound 13 (1.9g, 3.83 mmol) was added. The solution was stirred further for 30 min atroom temperature and concentrated. The residue was partitioned betweenethyl acetate and pH 7.0 aqueous buffer. The organic phase was washedwith brine, dried (Na₂ SO₄), and concentrated. The residue was purifiedby silica gel chromatography (Et₂ O) to afford 718 mg (36%) of the titleproduct.

¹ H NMR (CDCl₃) δ: 7.40-7.20 (10H, m), 5.14 (2H, s), 5.07 (2H, s), 4.43(1H, d, J=1.5), 3.50-3.40 (2H, m), 3.13-3.05 (1H, m), 2.57 (2H, q,J=7.7), 1.85-1.60 (4H, m), 1.23 (3H, t, J=7.7);

IR (film): 1764, 1738 cm⁻¹ ;

High resolution FAB MS Calcd. for C₂₅ H₃₁ N₄ O₅ S: 499.2015. Found:499.2010.

EXAMPLE 24trans-4-Ethylthio-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-acetyl-2-azetidinone(18)

To a -78° C. THF (10 mL) solution of compound 17 (718 mg, 1.26 mmol) wasadded 1N THF solution (1.5 mL) of lithium bis(trimethylsilyl)amide (1.5mmol), followed by stirring the solution for 10 min. Acetyl chloride(0.1 mL, 1.5 mmol) was added, and the resultant solution was warmed toroom temperature, followed by an additional hour of stirring. Thereaction mixture was concentrated, and the residue was partitionedbetween pH 7.0 aqueous buffer and ethyl acetate. The organic phase waswashed with brine, dried (Na₂ SO₄), and concentrated. The residue waspurified by silica gel chromatography (1:1/Et₂ O:hexane) to afford 615mg (90%) of the title product.

¹ H NMR (CDCl₃) δ: 7.42-7.22 (10H, m), 5.16 (2H, s), 5.08 (2H, s), 4.75(1H, d, J=2.3), 3.52-3.40 (2H, m), 3.07-2.84 (3H, m), 2.33 (3H, s),1.90-1.65 (4H, m);

High resolution FAB MS Calcd. for C₂₇ H₃₃ N₄ O₆ S: (M+H) 541.2121.Found: 541.2114.

EXAMPLE 25 trans-4-Ethylthio-3-guanidinopropyl-1-acetyl-2-azetidinonehydrochloride salt (VII)

A methanol/ethyl acetate (1.5 mL/1.5 mL) solution of compound 18 (117mg, 0.19 mmol) and 1N HCl (0.19 mL), containing 10% palladium on carbon,was stirred under a hydrogen atmosphere until TLC indicated thedisappearance of the starting material (about 1 h). The suspension wasfiltered through a pad of Celite, and the filtrate was concentrated toafford 40 mg (68%) of the title product as a foam.

¹ H NMR (CD₃ OD) δ: 4.93 (1H, d, J=3.6), 3.40-2.82 (5H, m), 2.32 (3H,s), 1.95-1.63 (4H, m), 1.27 (3H, t, J=7.4);

IR (film): 1795, 1735, 1713 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₁ H₂₁ N₄ O₂ S: (M+H) 273.1385.Found: 273.1391.

EXAMPLE 26 3-[N',N"-di(Cbz)guanidino]propyl-2-azetidinone (19 )

A dioxane (100 mL) solution of compound 17 (1.8 g, 3.5 mmol) containingRaney-Nickel (Aldrich) was stirred and heated for 30 min at 60° C. TheRaney-Nickel was filtered, and the filtrate was concentrated. Theresidue was purified by silica gel chromatography to afford 830 mg (45%)of the title product.

¹ H NMR (CDCl₃) δ: 7.38-7.17 (10H, m), 5.13 (2H, s), 5.05 (2H, s),3.47-3.35 (2H, m), 3.33 (1H, t, J=5.4), 3.23-3.14 (1H, m), 3.94-3.88(1H, m), 1.80-1.57 (4H, m);

IR (film): 1755 (shoulder), 1740 cm⁻¹ ;

High resolution FAB MS Calcd. for C₂₃ H₂₇ N₄ O₅ : (M+H) 439.1981. Found:439.1976.

EXAMPLE 27 3-[N',N"-di(Cbz)guanidino]propyl-1-acetyl-2-azetidinone (20)

To a -78° C. THF (6 mL) solution of compound 19 (366 mg, 0.84 mmol) wasadded 1N THF solution of lithium bis(trimethylsilyl)amide (1 mL, 1mmol), followed by 10 min of stirring. Acetyl chloride (0.071 mL, 1mmol) was added, and the solution was warmed to room temperature,followed by 1 h of stirring. The solution was partitioned between etherand pH 7.0 aqueous buffer. The organic phase was dried (Na₂ SO₄) andconcentrated. The residue was purified by silica gel chromatography(9:1/EtO₂ :hexane) to afford 255 mg (63%) of the title product.

¹ H NMR (CDCl₃) δ: 7.40-7.18 (10H, m), 5.14 (3/2H, s), 5.12 (1/2H, s),5.07 (3/2H, s), 5.05 (1/2H, s), 3.70-3.60 (1H, m), 3.48-3.35 (2H, m),3.30-3.15 (2H, m), 2.33 (9/4H, s), 2.32 (3/4H, s), 1.96-1.63 (4H, m);

IR (film): 1792, 1740, 1710 cm⁻¹ ;

High res. FAB MS Calcd. for C₂₅ H₂₉ N₄ O₆ : (M+H) 481.2087. Found:481.2076.

EXAMPLE 28 3-Guanidinopropyl-1-acetyl-2-azetidinone hydrochloride salt(VIII)

A methanol/ethyl acetate (2.5 mL/2.5 mL) solution of compound 20 (255mg, 0.58 mmol) and 1N HCl (0.58 mL), containing 10% palladium on carbon,was stirred under a hydrogen atmosphere until TLC indicated thedisappearance of the starting material (about 10 min). The suspensionwas filtered through a pad of Celite, and the filtrate was concentratedto afford 99 mg (69%) of the title compound as a yellow foam.

¹ H NMR (CD₃ OD) δ: 3.70 (1H, t, J=6.7), 3.40-3.16 (4H, m), 2.31 (3H,s), 1.87-1.65 (4H, m);

IR (film): 1790, 1700 (shoulder), 1670 cm⁻¹ ;

High resolution FAB MS Calcd. for C₈ H₁₇ N₄ O₂ : (M+H) 213.1352. Found:213.1349.

EXAMPLE 293-[3-[N',N"-di(Cbz)guanidino]propyl]-1-[(5-dimethylamino)-1-naphthalenesulfonyl]-2-azetidinone(21)

To a -78° C. THF (5 mL) solution of compound 19 (650 mg, 1.48 mmol) wasadded 1N THF solution of lithium bis(trimethylsilyl)amide (1 mL, 1mmol), followed by 10 min of stirring. Dansyl chloride (399 mg, 1.48mmol) was added. The resultant solution was warmed to room temperature,stirred for 1 h, and concentrated. The residue was partitioned betweenethyl acetate and pH 7.0 aqueous buffer. The organic phase was washedwith brine, dried (Na₂ SO₄), and concentrated. The residue was purifiedby silica gel chromatography (4:1/Et₂ O:hexane) to afford 186 mg (19%)of the title compound.

¹ H NMR (CDCl₃) δ: 8.56 (1H, d, J=9.2), 8.33 (1H, d, J=9.2), 8.28 (1H,d, J=6.9), 7.57-7.47 (2H, m), 7.35-7.20 (10H, m), 5.13 (2H, s), 5.05(2H, s), 3.67-3.59 (1H, m), 3.34-3.16 (3H, m), 2.80 (6H, s), 1.70-1.47(4H, m);

IR (KBr): 1798, 1738 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₇ H₃₈ N₅ O₇ S: (M+H) 672.2492.Found: 672.2479.

EXAMPLE 303-Guanidinopropyl-1-[(5-dimethylamino)-1-naphthanesulfonyl)]-2-azetidinonehydrochloride salt (IX)

A methanol/ethyl acetate (2.5 mL/2.5 mL) solution of compound 21 (186mg, 0.28 mmol) and 1N HCl (0.28 mL), containing 10% palladium on carbon,was stirred under a hydrogen atmosphere until TLC indicated thedisappearance of the starting material. The suspension was filteredthrough a pad of Celite, and the filtrate was concentrated to afford 90mg (73%) of the title product.

¹ H NMR (CD₃ OD) δ: 8.59 (1H, d, J=7.7), 8.45 (1H, d, J=7.6) 8.27 (1H d,J=7.4) 7.68-7.57 (2H m), 7.41 (1H, d, J=7.5), 3.64 (1H, t, J=5.9),3.31-2.99 (3H, m), 2.93 (6H s) 1.61-1.43 (4H, m);

IR (film): 1795 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₉ H₂₆ N₅ O₅ S: (M+H): 404.1756.Found: 404.1752.

EXAMPLE 31trans-4-Ethylsulfonyl-3-[3-[N,N"-di(Cbz)guanidino]propyl]-1-acetyl-2-azetidinone(22)

To a methylene chloride (5 mL) solution of compound 18 (469 mg, 0.77mmol) was added MCPBA (85% pure, 500 mg, 1.93 mmol) in one portion(exothermic). The resultant suspension was stirred for 30 min andpartitioned between more methylene chloride and aqueous saturated sodiumbicarbonate solution. The organic layer was dried (Na₂ SO₄) andconcentrated. The residue was purified by silica gel chromatography toafford 386 mg (88%) of the title product.

¹ H NMR (CDCl₃) δ: 7.42-7.30 (10H, m), 5.16 (2H, s), 5.10 (2H, ABquartet), 4.93 (1H, d J=2.3) 3.80-3.70 (1H, m), 3.62-3.18 (4H, m), 2.40(3H, s), 1.98-1.67 (4H, m), 1.38 (3H, t, J=8.4);

IR (KBr): 1780, 1700 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₁ H₂₁ N₄ O₄ S: (M+H) 305.1284.Found: 305.1276.

EXAMPLE 32trans-4-Ethylsulfonyl-3-guanidinopropyl-1-acetyl-2-azetidinonehydrochloride salt (X)

A methanol/ethyl acetate (2.5 mL/2.5 mL) solution of compound 22 (380mg, 0.66 mmol) and 1N HCl (0.66 mL), containing 10% palladium on carbon,was stirred under a hydrogen atmosphere until TLC indicated thedisappearance of the starting material. The suspension was filteredthrough a pad of Celite, and the solution was concentrated to afford 140mg (62%) of the product as a yellow foam.

¹ H NMR (CD₃ OD) δ: 5.27 (1H, d, J=3.1), 3.76-3.70 (1H, m), 3.49-3.19(4H, m), 2.39 (3H, s), 2.02-1.73 (1H, m), 1.42 (3H, t, J=7.4);

IR (film): 1810, 1720 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₁ H₂₁ N₄ O₄ S: (M+H) 305.1284.Found: 305.1276.

EXAMPLE 33trans-4-(1-Piperidinocarbonyl)-3-[3-[N',N"-di(Cbz)-guanidino]-propyl]-2-azetidinone(23)

To a THF (20 mL) solution of compound 6a (1.0 g, 2.1 mmol) was addedcarbonyldiimidazole (400 mg, 2.5 mmol), followed by stirring thesolution for 30 min. Piperidine (0.21 g, 2.5 mmol) was added, theresultant solution was further stirred for 1 h, and concentrated. Theresidue was partitioned between EtOAc and 1N HCl. The organic phase waswashed with brine, dried (MgSO₄), and concentrated. The residue waspurified by silica gel chromatography (100% EtOAc) to afford 720 mg(62%) of the title product.

¹ H NMR (CDCl₃) δ: 8.32 (1H, br t, J=5.1), 7.32-7.10 (10H, m), 5.07 (2H,s), 5.01 (2H, s), 3.93 (1H, s), 3.55-3.05 (7H, m), 1.85-1.30 (10H, m);

High resolution FAB MS Calcd. for C₂₉ H₃₆ H₅ O₆ : (M+H) 550.2666. Found:550.2656.

EXAMPLE 34trans-4-(1-Piperidinocarbonyl)-3-[3-[N',N"-di(Cbz)-guanidino]-propyl]-1-acetyl-2-azetidinone (24)

To a -78° C. THF (10 mL) solution of 23 (334 mg, 0.61 mmol) was added 1NTHF solution of sodium bis(trimethylsilyl)amide (0.65 mL, 0.65 mmol).The solution was stirred for 15 min, followed by the addition of acetylchloride (47.4 mg, 0.6 mmol). The resultant reaction mixture was broughtto room temperature and subsequently stirred for 30 min. The solutionwas concentrated, and the residue was partitioned between pH 4.0 aqueousbuffer and EtOAc. The organic layer was dried (MgSO₄) and concentrated.The residue was purified by silica gel chromatography (3:1/Et₂ O: EtOAc)to afford 155 mg (78%) of the title product.

¹ H NMR δ: 8.33 (1H, br t, J=5.3) 7.36-7.18 (10H, m), 5.13 (2H, s), 5.04(2H, s), 4.43 (1H, br s), 3.62-3.30 (6H, m), 3.28-3.18 (1H, m), 2.33(3H, s), 1.85-1.30 (10H, m);

IR (film): 1795, 1735, 1720 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₁ H₃₈ N₅ O₇ : (M+H) 592.2771. Found:592.2765.

EXAMPLE 35trans-4-(1-Piperidinocarbonyl)-3-guanidinopropyl-1-acetyl-2-azetidinonehydrochloride salt (XI)

A methanolic (3 mL) solution of compound 24 (150 mg, 0.27 mmol) and 1NHCl (0.27 mL), containing 10% palladium on carbon, was stirred under ahydrogen atmosphere for 5 min. The suspension was filtered through a padof Celite, and the filtrate was concentrated to afford 66 mg (68%) ofthe title compound.

¹ H NMR (D₂ O) δ: 4.93 (1H, br s), 3.76-3.22 (7H, m), 2.38 (3H, s),1.97-1.23 (10H, m);

IR (film): 1795, 1710 cm⁻¹ :

High resolution FAB MS Calcd. for C₁₅ H₂₆ O₃ N₅ : 324.2035. Found:324.2030.

EXAMPLE 36trans-4-(1-Piperidinocarbonyl)-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-[(5-dimethylamino)-1-naphthalenesulfonyl]-2-azetidinone (25)

To a -78° C. (12 mL) THF solution of compound 23 (640 mg, 1.16 mmol) wasadded 1N THF solution of sodium (1.25 mL, 1.25 mmol), followed bystirring the solution for 20 min. THF (20 mL) solution of dansylchloride (320 mg, 1.17 mmol) was added. The resultant solution wasbrought to room temperature and stirred for 45 min. The solution wasconcentrated, and the residue was partitioned between ethyl acetate andpH 4.0 aqueous buffer. The organic phase was dried (MgSO₄) andconcentrated. The crude product was purified by silica gelchromatography (100% Et₂ O) to afford 380 mg (42%) of the title product.

¹ H NMR (CDCl₃) δ: 8.57 (1H, d, J=7.1), 8.45 (1H, d, J=7.2), 8.26 (1H,d, J=6.0), 7.56 (1H, t, J=6.7), 7.51 (1H, t, J=6.7), 7.35-7.26 (10H, m),7.15 (1H, d, J=6.3), 5.16 (2H, s), 5.09 (2H, s), 4.68 (1H, d, J=1.6),3.55-3.27 (7H, m), 2.84 (6H, s), 1.87-1.35 (10H, m);

IR (KBr): 1800, 1737 cm⁻¹ ;

High resolution FAB MS Calcd. for C₄₁ H₄₇ N₆ O₈ S₁ : (M+H) 783.3176.Found: 783.3162.

EXAMPLE 37trans-4-(1-Piperidinocarbonyl)-3-guanidinopropyl-1-[(5-dimethylamino)-1-naphthalenesulfonyl]-2-azetidinone hydrochloride salt (XII).

A methanol/ethyl acetate (3 mL/1.5 mL) solution of compound 25 (360 mg,0.48 mmol) and 1N HCl (0.55 mL), containing 10% palladium on carbon, wasstirred under a hydrogen atmosphere for 10 min. The suspension wasfiltered through a pad of Celite, and the filtrate was concentrated toafford 196 mg (73%) of the title product.

¹ H NMR (CD₃ OD) δ: 8.82 (1H, br, d, J=7.0), 8.66 (1H, d. J=7.1), 8.36(1H, d, J=6.1), 7.81-7.76 (3H, m), 5.07 (1H, d, J=2.1), 3.72-3.50 (3H,m), 3.42-3.06 (4H, m), 3.22 (6H, s), 1.92-1.43 (10H, m);

IR (KBr): 1800, 1735 cm⁻¹ ;

High resolution FAB MS Calcd. for C₂₅ H₃₅ N₆ O₄ : (M+H) 515.2441. Found:515.2438.

EXAMPLE 38trans-4-(1-Piperidinocarbonyl)-3-[3-[N',N"-di(Cbz)-guanidino]-propyl]-1-p-toluenesulfonyl-2-azetidinone (26)

To a -78° C. THF (10 mL) solution of compound 23 (379 mg, 0.69 mmol) wasadded 1N THF solution of sodium bis(trimethylsilyl)amide (0.7 mL),followed by stirring the solution for 20 min. p-Toluenesulfonyl chloride(131 mg, 0.69 mmol) was added, and the resultant solution was brought toroom temperature. The solution was concentrated, and the residue waspartitioned between ethyl acetate and pH 4.0 aqueous buffer. The organicphase was dried (MgSO₄) and concentrated. The residue was purified bysilica gel chromatography to afford 160 mg (33%) of the title product.

¹ H NMR (CDCl₃) δ: 7.87 (2H, d, J=8.4) 7.28 (2H, d, J=8.4), 7.40-7.22(10H, m), 5.14 (2H, s), 5.07 (2H, s), 4.70 (1H, d, J=1.5 ), 3.54-3.32(6H, m), 3.23-3.15 (1H, m), 2.37 (3H, s), 1.85-1.40 (10H, m);

IR (KBr): 1802, 1740 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₆ H₄₂ N₅ O₈ S: (M+H) 704.275. Found:704.2767.

EXAMPLE 39trans-4-(1-Piperidinocarbonyl)-3-guanidinopropyl-1-p-toluenesulfonyl-2-azetidinonehydrochloride salt (XIII)

An ethyl acetate/methanol (2.5 mL/2.5 mL) solution of compound 26 (157mg, 0.22 mmol) and 1N HCl (0.22 mL), containing 10% palladium on carbon,was stirred under a hydrogen atmosphere for 10 minutes. The suspensionwas filtered through a pad of Celite and the filtrate was concentratedto afford 70 mg (67%) of the title compound.

¹ H NMR (CD₃ OD) δ: 7.86 (2H, d, J=8.3), 7.42 (2H, d, J=8.3), 5.06 (1H,d, J=2.6), 3.74-3.10, (7H, m), 2.44 (3H, s), 1.90-1.50 (10H, m);

IR (KBr): 1800, 1740 cm⁻¹ ;

High resolution FAB MS Calcd. for C₂₀ H₃₀ N₅ O₄ S: (M+H) 436.2018.Found: 436.2011.

EXAMPLE 40 trans-4-(2-Phenylethenyl)-3-[3-[N',N"-di(Cbz)guanidino]-propyl]-1-acetyl-2-azetidinone(27)

To a -78° C. THF (20 mL) solution of compound 3a (4.1 g, 7.64 mmol) wasadded 1N THF solution of sodium bis(trimethylsilyl)amide (7.64 mL),followed by stirring the solution for 15 min. Acetyl chloride (600 mg,7.6 mmol) was added. The solution was warmed to room temperature,stirred for 75 min, and partitioned between ether and pH 4.0 aqueousbuffer. The organic layer was dried (Na₂ SO₄) and concentrated. Theresidue was purified by silica gel chromatography (Et₂ O:hexane/3:1) toafford 2.3 g (52%) of the title product.

¹ H NMR (CDCl₃) δ: 8.36 (1H, br s), 7.43-7.20 (15H, m), 6.68 (1H, d,J=15.3), 6.20 (1H, dd, J=15.3, 7.7), 3.53-3.40 (2H, m), 3.10-3.0 (1H,m), 2.38 (3H, s), 1.90-1.65 (4H, m);

IR (film): 1780, 1735, 1710 cm⁻¹ ;

Anal. Calcd. for C₃₃ H₃₄ N₄ O₆ : C, 68.03; H, 5.88; N, 9.62. Found: C,68.05; H, 5.99; N, 9.30.

High resolution FAB MS Calcd. for C₃₃ H₃₅ N₄ O₆ : (M+H) 583.2556. Found:583.2543.

EXAMPLE 41trans-4-Formyl-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-acetyl-2-azetidinone(28)

Ozone was passed through a -78° C. methylene chloride solution ofcompound 27 (0.7 g, 1.2 mmol) until a blue color was maintained. Excessozone was removed by purging the solution with N₂. Dimethyl sulfide (1mL) was added. The solution was warmed to room temperature, allowed tostand for 72 h, and concentrated. The residue was purified by silica gelchromatography (EtOAc:Et₂ O/1:3) to afford 230 mg (38%) of the titleproduct.

¹ H NMR (CDCl₃) δ: 9.70 (1H, s ), 7.50-7.20 (10H, m), 5.17 (2H, s), 5.10(2H, s), 4.17 (1H, br s), 3.56-3.30 (2H, m), 3.30-3.20 (1H, m), 2.40(3H, s), 1.94-1.50 (4H, m);

IR (film): 1797, 1734, 1703 cm⁻¹ ;

High resolution FAB MS Calcd. for C₂₆ H₂₉ N₄ O₇ : (M+H) 509.2036. Found:509.2031.

EXAMPLE 42trans-4-Carboxy-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-acetyl-2-azetidinone(29)

To an acetone solution of compound 28 (3.5 g, 6.8 mmol) was addedfreshly prepared Jones reagent dropwise until TLC indicated thedisappearance of the starting material. The acetone layer was separated,and the residue was washed with a fresh portion of acetone. Two acetoneportions were combined and concentrated. The residue was purified bysilica gel chromatography (EtOAc:MeOH/99:1) to afford 2.4 g (66%) of thetitle product.

¹ H NMR (CDCl₃) δ: 7.40-7.11 (10H, m), 5.17 (2H, s), 5.11 (2H, s), 4.13(1H, d, J=2.3), 3.50-3.39 (2H, m), 3.26-3.17 (1H, m), 2.40 (3H, s),1.96-1.65 (4H, m);

High resolution FAB MS Calcd. for C₂₆ H₂₉ N₄ O₈ : 525.1985. Found:525.1991.

EXAMPLE 43 trans-4-Carboxy-3-guanidinopropyl-1-acetyl-2-azetidinonehydrochloride salt (XIV)

A methanol/ethyl acetate (3 mL/0.5 mL) solution of compound 29 (200 mg,0.38 mmol) and 1N HCl (0.5 mL), containing 10% palladium on carbon, wasstirred under a hydrogen atmosphere until TLC indicated thedisappearance of the starting material (15 min). The suspension wasfiltered through a pad of Celite, and the filtrate was concentrated toafford 37 mg (33%) of the title compound.

¹ H NMR (CD₃ OD) δ: 4.18 (1H, d, J=3.3), 3.37-3.20 (3H, m), 2.35 (3H,s), 2.02-1.74 (4H, m);

IR (KBr): 1800, 1709 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₀ H₁₉ N₄ O₄ : (M+H) 257.1250. Found:257.1244.

EXAMPLE 44trans-4-(2-Phenylethyl)-3-guanidinopropyl-1-acetyl-2-azetidinonehydrochloride salt (XV)

A methanol/ethyl acetate (3 mL/1 mL) solution of compound 27 (470 mg,0.82 mmol) and 1N HCl (0.9 mL), containing 10% palladium on carbon, wasstirred under a hydrogen atmosphere until TLC indicated thedisappearance of the starting material (about 30 min). The suspensionwas filtered through a pad of Celite, and the filtrate was concentratedto afford 220 mg (76%) of the title product.

¹ H NMR (CD₃ OD), selected peaks) δ: 7.5-7.10 (5H, m), 3.87-3.72 (1H,m), 2.30 (3H, s);

IR (film): 1770, 1710 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₇ H₂₅ N₄ O₂ : (M+H) 317.1977. Found:317.1971.

EXAMPLE 45trans-4-(4-Methylbenzeneaminocarbonyl)-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-acetyl-2-azetidinone(30)

To a -5° C. DMF (3.5 mL) solution of compound 29 (700 mg, 1.34 mmol) andp-toluidene (210 mg, 2 mmol) was added diphenylphosphoryl azide (550 mg,2 mmol). The resulting solution was stirred for 30 min, subsequentlywarmed to room temperature, and stirred for an additional 1.5 h. Thesolution was partitioned between Et₂ O and H₂ O. The organic layer wasdried (MgSO₄) and concentrated. The residue was chromatographed bysilica gel chromatography (Et₂ O:hexane/2:1) to afford 150 mg (18%) ofthe title product.

¹ H NMR (CDCl₃) δ: 7.40-7.20 (10H, m), 7.24 (2H, d, J=8.4), 7.03 (2H, d,J=8.4), 5.17 (2H, s), 5.07 (2H, AB quartet), 4.43 (1H, d, J=2.3),3.70-3.27 (3H, m), 2.42 (3H, s), 2.28 (3H, s), 2.0-1.62 (4H, m);

High resolution FAB MS Calcd. for C₃₃ H₃₆ N₅ O₇ : (M+H) 614.2615. Found:614.2602.

EXAMPLE 46trans-4-(4-Methylbenzeneaminocarbonyl)-3-guanidinopropyl-1-acetyl-2-azetidinonehydrochloride salt (XVI)

A methanol/ethyl acetate (2.0 mL/0.5 mL) solution of compound 30 (150mg, 0.25 mmol) and 1N HCl (0.28 mL), containing 10% palladium on carbon,was stirred under a hydrogen atmosphere until TLC indicated thedisappearance of the starting material (10 min). The suspension wasfiltered through a pad of Celite and the filtrate was concentrated toafford 70 mg (73%) of the title product.

¹ H NMR (CD₃ OD) δ: 7.45 (2H, d, J=7.2), 7.12 (2H, J=7.2), 4.32 (1H, d,J=3.2), 3.39-3.2 (3H, m), 2.37 (3H, s), 2.29 (3H, s), 1.91-1.59 (4H, m);

IR (KBr): 1802 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₇ H₂₄ N₅ O₃ : (M+H) 346.1879. Found:346.1875.

EXAMPLE 47 6-[N',N"-Di(Cbz)guanidino]hexanoic acid (31)

A methanolic (350 mL) solution of N,N'-dicarbobenzyloxy-S-methyl-isothiourea (60 g, 0.18 mol),triethylamine(18.2 g, 0.18 mol), and 6-aminocaproic acid (23.3 g, 0.18mol) was refluxed for 30 min and subsequently stirred at roomtemperature for 4 h. The solution was concentrated, and the residue waspartitioned between ethyl acetate and water. The organic phase wasseparated, dried (MgSO₄), and concentrated. The residual solid wasrecrystallized from ethyl acetate/hexane to afford 52.3 g (66%) of thetitle product, mp 93° C.

¹ H NMR (CDCl₃) δ: 7.43-7.20 (10H, m), 5.17 (2H, s), 5.10 (2H, s),3.47-3.34 (2H, m), 2.33 (2H, d, J=6.9), 1.71-1.50 (4H, m), 1.45-1.30(2H, m);

IR (KBr): 1740 cm⁻¹ ;

Anal. Calcd. for C₂₃ H₂₇ N₃ O₆ : C, 61.81; H, 5.89; N, 9.52 Found: C,61.39; H, 6.29; N, 9.65.

EXAMPLE 48

6-[N',N"-Di(Cbz)guanidino)hexanoic acid methyl ester (32)

To a THF (300 mL) solution of compound 31 (50.8 g, 0.12 mol) was addedcarbonyldiimidazole (21.4 g, 0.132 mol). The resultant suspension wasstirred for 1 h at room temperature. Methanol (75 mL) was added,followed by an additional stirring of the solution for 19 h. Thesolution was concentrated, and the residue was partitioned between waterand ether. The organic phase was dried (MgSO₄) and concentrated. Theresidue was purified by silica gel chromatography (Et₂ O:hexane/1:1) toafford 22.8 g (42%) of the title compound.

¹ H NMR (CDCl₃) δ: 7.42-7.20 (10H, m), 5.16 (2H, s), 5.12 (2H, s), 3.64(3H, s), 3.38 (2H, q, J=6.1), 2.27 (2H, t, J=6.9), 1.68-1.50 (4H, m),1.42-1.28 (2H, m);

IR (film): 1740 cm⁻¹ ;

Anal. Calcd. for C₂₄ H₂₉ N₃ O₆ : C, 63.28; H, 6.42; N, 9.23. Found: C,63.30; H, 6.51; N, 9.26.

EXAMPLE 49trans-4-(2-Phenylethenyl)-3-[4-[N',N"-di(Cbz)guanidino]butyl]-2-azetidinone(33)

To a -40° C. THF (15 mL) solution of hexamethyldisilazane was added 2.5MTHF solution of n-BuLi (60 mL, 0.15 mol). The resultant solution wasstirred at -40° C. for 1 h and cooled to -78° C. A THF (30 mL) solutionof compound 32 (22.2 g, 0.05 mol) was added, followed by stirring thesolution for 1 h. The reaction mixture was warmed to -30° C. and stirredfor 1 h. The solution was subsequently cooled to -78° C. A THF (30 mL)solution of N-(trimethylsilyl)benzaldimine (10.2 g, 0.05 mol) was added,followed by warming the solution to -30° C. and stirring for 30 min. Thesolution was warmed to -20° C. and allowed to stand at this temperaturefor 16 h. The resultant dark reaction mixture was concentrated to halfof its original volume and partitioned between ether and 1N HCl; enoughacid was used to eventually maintain the pH of the aqueous phase below2.0. The organic phase was washed with water and brine, dried (MgSO₄),and concentrated. The residue was purified by silica gel chromatography(Et₂ O:ETOAc/9:1) to afford 6.8 g (36%) of the title product.

¹ H NMR (CDCl₃) δ: 7.43-7.20 (15H, m), 6.58 (1H, d, J=15.3), 6.18 (1H,dd, J=15.3, 8.4), 5.17 (2H, s), 5.10 (2H, s), 3.93 (1H, d, J=8.4, 2.0),3.48-3.37 (2H, q, J=6.6), 2.95 (1H, br t, J=6.6), 1.92-1.40 (6H, m);

IR (KBr): 1760, 17333 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₂ H₃₅ N₄ O₅ : (M+H) 555.2607. Found:555.259.

EXAMPLE 50trans-4-(2-Phenylethenyl)-3-[4-[N',N"-di(Cbz)-guanidino]butyl]-1-t-butyldimethylsilyl-2-azetidinone(34)

To a 0° C. DMF (25 mL) solution of compound 33 (6.8 g, 12.3 mmol) wasadded triethylamine (2.05 g, 20 mmol) and tert-butyldimethylsilylchloride (3.05 g, 20 mmol). The reaction mixture was warmed to roomtemperature and stirred for 1.5 h. The suspension was partitionedbetween Et₂ O and pH 7.0 aqueous buffer. The organic layer was dried(MgSO₄) and concentrated. The residue was purified by silica gelchromatography to afford 8.4 g (quantitative) of the title product.

¹ H NMR (CDCl₃) δ: 7.40-7.15 (15H, m), 6.52 (1H, d, J=16.8), 6.1 (1H, d,J=16.8, 8.4), 5.1 (2H, s), 5.05 (2H, s), 3.77 (1H, d, J=8.4, 2.0), 3.37(2H, q, J=6.1), 2.95-2.87 (1H, m), 1.85-1.35 (6H, m), 0.9 (9H, s), 0.2(3H, s), 0.1 (3H, s);

IR (film): 1735 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₈ H₄₉ N₄ O₅ Si: (M+H) 669.3472.Found: 669.3466.

EXAMPLE 51 Step Atrans-4-Formyl-3-[4-[N',N"-di(Cbz)quanidino]butyl]-1-t-butyldimethylsilyl-2-azetidinone(35)

Ozone was passed through a -78° C. methylene chloride (100 mL) solutionof compound 34 (8.2 g, 12 mmol) until the solution retained a bluecolor. Dimethyl sulfide (10 mL) was added. The solution was warmed toroom temperature and allowed to stand for 16 h. The solution wasconcentrated to afford the title product which was not purified but useddirectly in Step B.

Step Btrans-1-Methylcarbonyl-3-[4-[N',N"-di(Cbz)quanidino]butyl]-2-azetidinone(36)

The aldehyde prepared in Step A was dissolved in acetone (100 mL). Tothe resultant solution was added Jones reagent until TLC indicated thedisappearance of starting material. The resultant solution was stirredfor 2 h, concentrated to one third of its original volume, andpartitioned between ethyl acetate and water. The organic layer wasseparated, dried (MgSO₄), and concentrated. The residue was elutedthrough silica gel with ethyl acetate: acetic acid (99:1) to afford 0.6g of dark oil which was dissolved in methylene chloride. An ethersolution of diazomethane was added dropwise until yellow colorpersisted. Excess diazomethane was decomposed with acetic acid. Thesolution was concentrated. The residue was purified by silica gelchromatography (Et₂ O) to afford 250 mg (24% overall from compound 36)of the title product.

¹ H NMR (CDCl₃) δ: 7.38-7.20 (10H, m), 5.13 (2H, s ), 5.07 (2H, s), 3.38(2H, q, J=6.1), 3.17 (1H, br t, J=9.0), 1.89-1.37 (6H, m);

High resolution FAB MS Calcd. for C₂₆ H₃₁ N₄ O₇ : (M+H) 511.2193. Found:511.2182.

EXAMPLE 52trans-4-Methoxycarbonyl-3-[4-[N',N"-di(Cbz)guanidino]butyl]-1-[(5-dimethylamino)-1-naphthalenesulfonyl]-2-azetidinone (37)

To a -78° C. THF (3 mL) solution of compound 36 (250 mg, 0.49 mmol) wasadded 1N THF solution of sodium bis(trimethylsilyl)amide (0.55 mL). Theresultant solution was stirred for 20 min. A THF (1 mL) solution ofdansyl chloride (0.135 g, 0.5 mmol) was added. The resultant solutionwas allowed to warm to room temperature and concentrated. The residuewas partitioned between ethyl acetate and aqueous pH 7.0 buffer. Theorganic layer was dried (MgSO₄) and concentrated. The residue waspurified by silica gel chromatography to afford 141 mg (39%) of thetitle product.

¹ H NMR (CDCl₃) δ: 8.59 (1H, d, J=8.5), 8.47 (1H, d, J=8.7), 8.27 (1H,d, J=6.2), 7.60 (1H, t, J=7.6), 7.53 (1H, t, J=7.5), 7.37-7.17 (11H, m),5.15 (2H, s), 5.09 (2H, s), 4.21 (1H, d, J=3.1), 3.45 (3H, s), 3.34-3.22(3H, m), 2.85 (6H, s), 1.81-1.36 (6H, m);

FAB MS: 744 (M+H).

EXAMPLE 53 trans-4-Methoxycarbonyl-3-(4-guanidinobutyl)-1-[(5-dimethylamino)-1-naphthalenesulfonyl]-2-azetidinone hydrochloride salt (XVII)

A methanol/ethyl acetate (3 mL/1 mL) of compound 37 (130 mg, 0.18 mmol)and 1N HCl, containing 10% palladium on carbon, was stirred under ahydrogen atmosphere for 16 h. The suspension was filtered through a padof Celite, and the filtrate was concentrated to afford 45 mg (50%) ofthe title product.

¹ H NMR (CD₃ OD) δ: 8.99 (1H, d, J=8.5), 8.86 (1H, d, J=8.2), 8.43 (1H,d, J=7.1), 8.10 (1H, d, J =6.9), 7.94-7.89 (2H, m), 4.52 (1H, s), 3.49(3H, s), 3.44 (6H, s), 3.33-3.03 (3H, m), 1.88-1.20 (6H, m);

IR (film): 1807, 1753 cm⁻¹ ;

High resolution FAB MS Calcd. for C₂₂ H₃₀ N₅ O₅ S: (M+H) 467.1968.Found: 476.1978.

EXAMPLE 54trans-4-[[2-[(Phenylmethoxy)carbonyl]-1-pyrrolidinyl]carbonyl]-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-acetyl-2-azetidinone (38)

To a -5° C. methylene chloride (1 mL) solution of ethyl chloroformate(0.12 g, 1.1 mmol) was added a methylene chloride solution of compound29 (0.35 g, 0.7 mmol), followed by the addition of triethylamine (0.112g, 1.1 mmol). The resultant reaction mixture was stirred for 1 h between-5° C. and 5° C. Triethylamine (0.11 g, 1.1 mmol) and prolinebenzylester hydrochloride (260 mg, 1.1 mmol) was added, and the solutionwas stirred between -5° C. to 5° C. for an additional 15 min and for 16h at room temperature. The solution was washed with saturated aqueousNaHCO₃ solution, dried (MgSO₄), and concentrated. The residue waspurified by silica gel chromatography (Et₂ O:EtOAc/8:2) to afford 70 mg(14%) of the title compound.

¹ H NMR (CDCl₃) δ: 7.40-7.13 (15 H, m), 5.18-5.03 (6 H, m), 4.60-4.43(1/2 H, m), 4.26-4.20 (1/2 H, m), 3.92-3.17 (6 H, m), 2.34 (3/2 H, s),2.29 (3/2 H, s), 2.24-1.50 (8 H, m);

High resolution FAB MS Calcd. for C₃₈ H₄₂ N₅ O₉ : (M+H) 712.2990. Found:712.2982.

EXAMPLE 55trans-4-[(2-Carboxy-1-pyrrolidinyl)carbonyl]-3-(3-guanidino)propyl-1-acetyl-2-azetidinonehydrochloride salt (XVIII)

A methanol/ethyl acetate solution of compound 38 (150 mg, 0.2 mmol) and1N HCl, containing 10% palladium on carbon, was stirred under a hydrogenatmosphere for 1.5 h. The suspension was filtered through a pad ofCelite, and the filtrate was concentrated to afford 47 mg (60%) of thetitle product.

¹ H NMR (CD₃ OD) δ: 4.59 (1/4 H, d, J=2.9), 4.52-4.49 (1/4H, m),4.45-4.40 (1/4 H, m), 4.33 (1/4 H, d, J=2.7 ), 4.04-3.92 (1/2 H, m),3.58-3.50 (1/2 H, m) 3.42-3.15 (5H, m), 2.33 (3/2 H, s), 2.32 (3/2 H,s), 2.13-1.63 (8H, m);

IR (KBr): 1800, 1712 cm⁻¹.

EXAMPLE 56trans-4-[[[2-Oxo-2-(phenylmethoxy)ethyl]amino]carbonyl]-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-acetyl-2-azetidinone(39)

To a -10° C. methylene chloride (2 mL) solution of ethyl chloroformate(57 mg, 0.53 mmol) was added a methylene chloride (2 mL) solution ofcompound 29 (220 mg, 0.44 mmol) and triethylamine (530 mg, 0.13 mmol).The resultant solution was stirred between -5° C. and 5° C. for 1 h.Glycine benzyl ester hydrochloride (O.11 g, 0.64) was added, followed bytriethylamine (0.53 g, 0.13 mmol). The resulting suspension was stirredat room temperature for 21 h and filtered through a sintered glassfunnel. The solid was washed with methylene chloride. The filtrate andthe washing portion were combined and washed with 1N HCl, dried (MgSO₄),and concentrated. The residue was purified by silica gel chromatography(Et₂ O:EtOAc/9:1) to afford 140 mg (47% of the title product.

¹ H NMR (CD₃ OD) δ: 7.43-7.18 (15H, m), 5.30-5.00 (6H, m), 4.26 (1H, d,J=3.2), 4.13 (1H, dd, J=18.1, 6.6), 3.82-3.63 (1H, m), 3.57 (1H, dd,J=18.1, 4.8), 3.38-3.28 (1H, m), 3.27-3.17 (1H, m), 2.39 (3H, s),2.0-1.5 (4H, m);

IR (film): 1798, 1738, 1715 cm⁻¹.

EXAMPLE 574-[[(2-Hydroxy-2-oxoethyl)amino]carbonyl]-3-(3-guanidinopropyl)-1-acetyl-2-azetidinonehydrochloride salt (XIX)

A methanol/ethyl acetate solution of compound 41 (240 mg, 0.36 mmol) and1N HCl (0.4 mL), containing 10% palladium on carbon, was stirred under ahydrogen atmosphere for 1 h. The suspension was filtered through a padof Celite, and the filtrate was concentrated to afford 55 mg (44%) ofthe title product.

1H NMR (CD₃ OD) δ: 4.24 (1H, d, J=3.3), 4.08-3.85 (2H, m), 3.38-3.13(3H, m), 2.35 (3H, s), 1.97-1.58 (4H, m);

IR (KBr): 1802, 1758, 1720, 1670 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₂ H₂₀ N₅ O₅ : (M+H) 314.1469. Found:314.1460.

EXAMPLE 584-Acetyloxy-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-acetyl-2-azetidinone(40)

To a -78° C. THF (20 mL) solution of compound 13 (trans:cis ratio, 2:1;580 mg; 1.16 mmol) was added 1N THF solution of sodiumbis(trimethylsilyl)amide (1.36 mL, 1.36 mmol), followed by 10 minstirring of the resultant solution. Acetyl chloride (96 mL, 1.36 mmol)was added, and the temperature of the resultant solution was raised toroom temperature, stirred for 30 min, and concentrated. The residue waspartitioned between ether and water. The organic phase was dried (Na₂SO₄) and concentrated. The residue was purified by silica gelchromatography (4:1/ether:hexane) to afford 420 mg (57%) of the productas a mixture of trans and cis (2:1) isomers.

¹ H NMR (CDCl₃) δ: 7.42-7.20 (10H, m), 6.82 (1/3 H, d, J=5.4), 6.04 (2/3H, d, J=1.5), 5.14 (2H, s), 5.08 (2H, s), 3.52-3.40 (2H, m), 3.17-3.07(1H, m), 2.36 (2H, s), 2.32 (1H, s), 2.08 (2H, s), 2.05 (1H, s),1.90-1.57 (4H, m);

IR (film): 1803, 1758, 1723 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₁ H₂₀ N₄ O₄ : (M+H) 271.1406. Found:271.1404.

EXAMPLE 59 4-Acetyloxy-3-guanidinopropyl-1-acetyl-2-azetidinonehydrochloride salt (XX)

A methanol/ethyl acetate (1:1/5 mL:5 mL) solution of compound 40 (420mg, 0.78 mmol) and 1N HCl (0.76 mL), containing 10% palladium on carbon,was shaken in a Parr hydrogenator at 45 psi hydrogen pressure for 2 h.The suspension was filtered through a pad of Celite, and the filtratewas concentrated to afford 170 mg (71%) of the title product as amixture of trans and cis (2:1) isomers.

¹ H NMR (CD₃ OD) δ: 6.55 (1/3 H, d, J=5.0), 6.11 (2/3 H, d, J=1.7),3.34-3.13 (3H, m), 2.33 (2H, s), 2.31 (1H, s), 2.11 (1H, s), 2.09 (2H,s), 1.95-1.63 (4H, m);

IR (film): 1805, 1760, 1720 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₁ H₁₉ N₄ O₄ : (M+H) 271.1406. Found:271.1404.

EXAMPLE 604-Acetyloxy-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-t-butylcarbonyl-2-azetidinone(41)

To a -78° C. THF (6 mL) solution of compound 13 (trans:cis ratio, 2:1;600 mg; 1.2 mmol) was added 1N THF solution of sodiumbis(trimethylsilyl)amide (1.2 mL, 1.2 mmol), and the resultant solutionwas stirred for 15 min. Pivaloyl chloride (150 mg, 1.24 mmol) was added,and the solution was stirred for 30 min at -78° C. and for 1.5 h at roomtemperature. The reaction mixture was partitioned between pH 4.0 aqueousbuffer and ether. The organic phase was dried (MgSO₄) and concentrated.The residue was purified by silica gel chromatography (1:1/Et₂ O:hexane)to afford 300 mg (43%) of the title product as a mixture of trans andcis (3:2) isomers.

¹ H NMR (CDCl₃) δ: 7.41-7.20 (10H, m), 6.53 (2.5 H, d, J=5.3), 6.07(3/5, d, J=1.5), 5.14 (2H, s), 5.08 (2H, s), 3.47-3.38 (2H, m),3.03-2.97 (1H, m), 2.06 (1.8 H, s), 2.04 (1.2 H, s), 1.84-1.60 (4H, m),1.28 (5.4 H, s), 1.26 (3.6 H, s);

IR (film): 1802, 1734, 1703 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₀ H₃₇ N₄ O₈ : 581.2611. Found:581.2603.

EXAMPLE 61 4-Acetyloxy-3-guanidinopropyl-1-t-butylcarbonyl-2-azetidinonehydrochloride salt (XXI)

A methanol/ethyl acetate solution of compound 41 (300 mg, 0.5 mmol) and1N HCl (0.52 mL), containing 10% palladium on carbon, was stirred undera hydrogen atmosphere for 30 min. The suspension was filtered through apad of Celite, and the filtrate was concentrated to afford 0.1 g (64%)of the title product as a mixture of trans and cis (3:2) isomers.

¹ H NMR (CD₃ OD) δ: 6.56 (2/5 H, d, J=5.1), 6.11 (3/5 H, d, J=1.6),3.35-3.10 (3H, m), 2.10 (1.2H, s), 2.08 (1.8H, s), 1.97-1.52 (4H, m),1.27 (5.4H, s), 1.26 (3.6H, s);

IR (KBr): 1804, 1751, 1705 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₄ H₂₅ N₄ O₄ : (M+H) 313.1876. Found:313.1872.

EXAMPLE 624-Acetyloxy-3-[3-[N',N"-di(Cbz)guanidino]propyl]-1-phenylcarbonyl-2-azetidinone(42)

To a -78° C. THF (4 mL) of compound 13 (500 mg, 1 mmol) was added 1N THFsolution of sodium bis(trimethylsilyl)amide (1.0 mL, 1.0 mmol), and theresultant reaction mixture was stirred for 15 min. Benzoyl chloride (140mg, 1 mmol) was added, and the mixture was stirred for 30 min at -78° C.and for 90 min at room temperature. The solution was subsequentlypartitioned between ether and aqueous pH 4.0 buffer. The organic layerwas dried (MgSO₄) and concentrated. The residue was purified by silicagel chromatography (Et₂ O:hexane/2:1) to afford 200 mg (33%) of thetitle product as a mixture of trans and cis (3:1) isomers.

¹ H NMR (CDCl₃) δ: 7.63-7.16 (15H, m), 6.75 (1/4 H, d, J=5.1), 6.30(3/4, d, J=1.9), 5.15 (2H, s), 5.10 (2H, s), 3.56-3.35 (2H, m),3.30-3.16 (1H, m), 2.11 (2.25 H, s), 2.09 (0.75 H, s), 1.93-1.63 (4H,m);

IR (film): 1866, 1749 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₂ H₃₃ N₄ O₈ : (M+H) 601.2298. Found:601.2290.

EXAMPLE 63 4-Acetyloxy-3-guanidinopropyl-1-phenylcarbonyl-2-azetidinonehydrochloride salt (XXII)

A methanol/ethyl acetate solution of compound 44 (200 mg, 0.33 mmol) and1N HCl (0.33 mL), containing 10% palladium on carbon, was stirred undera hydrogen atmosphere for 30 min. The suspension was filtered through apad of Celite, and the filtrate was concentrated to afford 100 mg (82%)of the title product as a mixture of trans and cis (3:1) isomers.

¹ H NMR (CD₃ OD) δ: 7.68-7.42 (5H, m), 6.76 (1/4 H, d, J=5.2), 6.32 (3/4H, d, J=1.9), 3.44-3.10 (3H, m), 2.14 (0.75H, s, 2.14), 3/4 2.12 (2.25H,s), 2.06-1.58 (4H, m);

IR (film): 1806, 1749 cm⁻¹ ;

High resolution FAB MS Calcd. for C₁₆ H₂₁ N₄ O₄ : (M+H) 333.1563. Found:333.1565.

EXAMPLE 64cis-4-(1-Piperidinocarbonyl)-3-[3-[N',N"-di(Cbz)-guanidino]propyl]-2-azetidinone(43)

To a THF (12 mL) solution of compound 6b (1.3 g, 2.7 mmol) was addedcarbonyldiimidazole (0.53 g, 3.24 mmol), and the resultant solution wasstirred for 35 min. Piperidine (230 mg, 2.7 mmol) was added, and thereaction mixture was stirred for an additional hour and concentrated.The residue was partitioned between EtOAc and 0.5N HCl. The organicphase was dried (MgSO₄) and concentrated. The residue was purified bysilica gel chromatography to afford 360 mg (24%) of the title compound;

¹ H NMR (CDCl₃) δ: 7.38-7.18 (10H, m), 5.12 (2H, s), 5.03 (2H, s), 4.33(1H, d, J=5.4), 3.52-3.02 (7H, m), 1.90-1.40 (10H, m).

EXAMPLE 65cis-4-(1-Piperidinocarbonyl)-3-[3-[N',N"-di(Cbz)-guanidino]propyl]-1-acetyl-2-azetidinone(44)

To a -78° C. THF (2 mL) solution of compound 43 (360 mg, 0.65 mmol) wasadded 1N THF solution of sodium bis(trimethylsilyl)amide (0.65 mL), andthe resultant solution was stirred for 20 min. Acetyl chloride (0.05 mL,0.7 mmol) was added, and the solution was warmed to room temperature andstirred for an additional hour. The solution was partitioned betweenether/ethyl acetate and pH 4.0 aqueous buffer solution. The organicphase was dried (MgSO₄) and concentrated. The residue was purified bysilica gel chromatography (100% EtOAc) to afford 240 mg (62%) of thetitle product.

¹ H NMR (CDCl₃) δ: 7.40-7.20 (10H, m), 5.13 (2H, s), 5.06 (2H, s), 6.76(1H, d, J=6.1), 3.58-3.12 (7H, m), 2.36 (3H, s), 1.86-1.34 (10H, m);

IR (KBr): 1798, 1740, 1722 cm⁻¹ ;

High resolution FAB MS Calcd. for C₃₁ H₃₈ N₅ O₇ : (M+H) 592.2711. Found:592.2769.

EXAMPLE 66cis-4-(1-Piperidinocarbonyl)-3-guanidinopropyl-1-acetyl-2-azetidinonehydrochloride salt (XXIII)

A methanol (2 mL) solution of compound 46 (240 mg, 0.4 mmol) and 1N HCl(0.5 mL), containing 10% palladium on carbon, was stirred under ahydrogen atmosphere for 15 min. The suspension was filtered through apad of Celite, and the filtrate was concentrated to afford 124 mg (86%)of the title product.

¹ H NMR (CD₃ OD) δ: 5.04 (1H, d, J=6.7), 3.83-3.17 (7H, m), 2.34 (3H,s), 192-1.50 (10H, m);

IR (film): 1798, 1715 cm⁻¹ :

High resolution FAB MS Calcd. for C₁₅ H₂₆ N₅ O₃ : (M+H) 324.2035. Found:324.2032.

EXAMPLE 67 N-(trimethylsilyl)cinnamilydenimine

A procedure analogous to that reported by C. Kruger et al. [Chem. Ber.96, 2132 (1963)] was employed. To a -78° C. THF (1600 ml) solution of1,1,1,3,3,3-hexamethyldisilazane (221.9 g, 137 mol) was added 2.5Mhexane solution of n-butyllithium (550 mL, 1.38 mole). The resultantsolution was stirred for 30 min. A THF (200 mL) solution ofcinnamaldehyde (165.2 g, 1.25 mole) was added dropwise, and the solutionwas stirred for another 30 min. Trimethylchlorosilane (149.4 g, 1.38mol) was added, and the solution was allowed to warm up to roomtemperature and stirred for 2 h. The suspension was concentrated. Theresidue was triturated with anhydrous ether, and the solid was separatedby passing the suspension through a pad of Celite. The filtrate wasconcentrated, and the residue was distilled under reduced pressure toafford 190 g (68%) of the title product, b.p. 85° C. (0.15 mmHg).

¹ H NMR (CDCl₃) δ: 8.56 (1H, d, J=7.6), 7.38-7.03 (5H, m), 6.94 (1H, d,J=16.1), 6.68 (1H, dd, J=16.1, 7.6), 0.11 (9H,S), 0.08(3H, S), 0.01 (3H,s).

EXAMPLE 68 Biological Testing 1. Enzyme Assays for the Inhibition ofThrombin

The following reagents were used in these assays:

Thrombin assay buffer: 145 mM NaCl, 5 mM KCl, 30 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, 1 mg/ml polyethylene glycol(PEG-8000).

3 mM D-Phe-Pip-Arg-p-nitroanilide (s-2238) in H₂ O.

3 U/ml purified human α-thrombin dissolved in thrombin assay buffer.

Inhibitors to be tested were dissolved in H₂ O, methanol, or DMSO justprior to use.

Assay Procedure

To each well in a 96-well microtiter plate, 270 μl of assay buffer wasadded. Human α-thrombin (10 μl of 3 U/ml) was added, then 10 μl ofinhibitor were added and mixed. The samples were incubated at roomtemperature for a defined period of time (3 min for initial IC₅₀determinations). The enzymic reaction was started with 10 μl of 3 mMs-2238 substrate and continued at room temperature. The change inoptical density was measured at 405 nm. A kinetic microplate reader(Molecular Devices Corporation V_(max)) was used to measure the changein optical density over time.

Results are reported in Table 1 as IC₅₀ values, i.e., the drugconcentration in mole/liter which caused 50% inhibition of the enzymeactivity (after incubation of the drug with the enzyme for 3 min).

Alternatively, results are also reported in Table 1 expressed as k₂/k_(i) which is the second order rate constant in per mole per minutefor inactivation time of the enzyme [Kitz, et al., J. Biol. Chem., 237,3245 (1962)].

2. Enzyme Assays for the Inhibition of Trypsin

The following reagents were used in these assays:

Trypsin assay buffer: 2 mM CaCl₂, 50 mM Tris/Cl pH 8.0.

3 mM Z-Val-Gly-Arg-pNA (Chromzyme TRY) dissolved in H₂ O.

6 μg/ml of purified bovine pancreatic trypsin dissolved in trypsin assaybuffer.

Inhibitors to be tested were dissolved in H₂ O, methanol or DMSO justprior to use.

Assay Procedure

To each well in a 96-well microtiter plate, 270 μl of assay buffer wasadded. Bovine trypsin (10 μl of 6 μg/ml) was added. Inhibitor (10 μl)was added, mixed, and then incubated at room temperature for 3 min. Theenzymic reaction was started with 10 μl of 3 mM Z-Val-Gly-Arg-pNAsubstrate. The change in optical density was measured at 405 nm at roomtemperature. A kinetic microplate reader (Molecular Devices CorporationV_(max)) was used to measure the change in optical density over time.

Results are reported in Table 1 as IC₅₀ values, i.e., the drugconcentration in mole/liter which caused 50% inhibition of the enzymeactivity (after incubation of the drug with the enzyme for 3 min).

Procedure for Determining the Concentration Required for DoublingThrombin Clotting Time--Clotting Time Assays

The following reagents were used in these assays:

Owren's Veronal Buffer: 125 mM NaCl, 28.4 mM sodium barbital, pH 7.35.

Human citrated plasma obtained from human volunteers or citrated plasmaobtained from IP-dosed animals (prepared as described below).

25 NIH Units/ml human α-thrombin in thrombin buffer for use with ratplasma.

10 NIH Units/ml human α-thrombin in thrombin buffer for use with humanplasma.

Preparation of the Citrated Plasma

Human Plasma: Blood from human volunteers was drawn into vacutainertubes containing one tenth final volume of 0.129M (3.8%) bufferedcitrate (16 mg Na₃ Citrate.2H₂ O and 2.1 mg citric acid per milliliterof H₂ O). The blood was centrifuged at 3500 rpm (480 x g) for 15 min atroom temperature (using a Sorvall RT 6000B centrifuge). The plasma wasremoved, pooled, and aliquoted into small tubes which were stored frozenfor later use.

Dosing: Test compound was prepared just prior to dosing. Routinely thedrugs are dissolved in water. Animals were dosed by i.p. injection.

Blood Drawing for Rats: After the appropriate time period, the animalswere ether-anesthetized, and blood was drawn by cardiac puncture using333 μl of 3.8% sodium citrate per 3 ml blood. After all of the sampleswere obtained, the tubes were centrifuged at 1,500 rpm for 15 min asdescribed for the human blood samples.

Clotting Time Measurement

Clotting times were determined by pipetting 0.1 ml of Owren's buffer(pre-warmed 37° C.) and 0.1 ml of human or rat plasma into yellow samplecuvettes. For studies with human plasma 10 U/ml human thrombin (10 ml)was placed in the reservoir assembly station of the MLA 700. (MedicalLaboratory Automation, Electra 700 Reservoir Assembly). For rat studies,the human thrombin concentration was 25 U/ml. The cuvettes were vortexedand then placed on the MLA 700 sample wheel. The coagulation timer (MLA700) automatically dispenses 0.1 ml human thrombin into the sample ineach cuvette. Detection of the fibrin clot was determined optically bythe MLA 700.

Studies were performed to determine the concentration of drug whichcaused a doubling of the clotting time in human plasma. From standardcurves of thrombin activity added to the sample versus the clottingtime, the concentration of drug which caused a doubling of the thrombinclotting time corresponded to inhibition of approximately 1/2 of theadded thrombin clotting activity. Results are reported in Table 1 asED₅₀ values, i.e., the drug concentration required to double clottingtime.

In separate studies, two compounds, Compound XIX and Compound XI, weredosed 50 mg/kg I.P. to three rats in each study group. Both compoundsprolonged clotting time as measured ex vivo (by the method describedabove). Results of such study are presented in Table 2. "Term" indicates"terminated sample" where a clot was unable to form after approximately99 seconds. When this occurred, the animal plasma was diluted 1:1 withcontrol rat plasma and the sample tested again. If needed, the animalplasma was diluted 1:4 and tested again.

The foregoing biological results show that the compounds of the presentinvention exhibit anti-thrombin and anti-tripsin activities and are thususeful in controlling blood coagulation and treating pancreatitis.

                                      TABLE 1                                     __________________________________________________________________________    BIOLOGICAL ACTIVITIES                                                         Example                                                                             Compound                                                                            Thrombin             Trypsin                                      Number                                                                              Number                                                                              k.sub.2 /k.sub.i (m.sup.-1 min.sup.-1)                                                 IC.sub.50 (nM)                                                                      ED.sub.50 (μM)                                                                   IC.sub.50 (nM)                               __________________________________________________________________________    10    I     1.2 × 10.sup.8                                                                   2-9   65    8                                            12    II    1.9 × 10.sup.7                                                                   2     1.25  40                                           14    III   2.1 × 10.sup.7                                                                   16    50    720                                          16    IV    1.5 × 10.sup.6                                                                   20    NT    70                                           18    V     8.3 × 10.sup.4                                                                   1700  30    5.0                                          22    VI    3.0 × 10.sup.6                                                                   3.2   15    750                                          25    VII   5.6 × 10.sup.6                                                                   12.5  0.75  36                                           28    VIII  2.5 × 10.sup.5                                                                   100   4.0   NT                                           30    IX    *        1000  30    NT                                           32    X     3.0 × 10.sup.6                                                                   NT    NT    NT                                           35    XI    4.2 × 10.sup.5                                                                   90    0.5   9                                            37    XII   2.8 × 10.sup.6                                                                   10    30    14                                           39    XIII  4.5 × 10.sup.7                                                                   3     80    120                                          43    XIV            390   12    12                                           44    XV    1.5 × 10.sup.6                                                                   210   3     470                                          46    XVI   1.3 × 10.sup.6                                                                   25    0.5   120                                          53    XVII  3.7 × 10.sup.5                                                                         50    NT                                           55    XVIII 4.3 × 10.sup.6                                                                   49    1.25  NT                                           57    XIX   2.0 × 10.sup.7                                                                   12    0.43  NT                                           59    XX    4.2 × 10.sup.7                                                                   12    0.34  12                                           61    XXI   2.4 × 10.sup.6                                                                   74    2.0   NT                                           63    XXII  *        35    10.0  NT                                           66    XXIII 4.3 × 10.sup.4                                                                   NT    20    NT                                           __________________________________________________________________________     *not measurable                                                               NT = not tested                                                          

                                      TABLE 2                                     __________________________________________________________________________                    Thrombin                                                                            Thrombin   Thrombin                                                     Induced                                                                             Induced    Induced                                                      Clotting                                                                            Clotting   Clotting                                                     Time  Time       Time                                         Animal                                                                            Condition   (Seconds)                                                                           (1:1 Control Plasma)                                                                     (1:4 Control Plasma)                         __________________________________________________________________________     1  Control     10.9                                                           2  No drug     11.1                                                           3              10.9                                                           4  Compound XIX 0.5 hr.                                                                      term. 14.4                                                     5  post dose   45.2                                                           6              term. 17.3                                                     7  Compound XIX 2 hr.                                                                        12.9                                                           8  post dose   13.3                                                           9              14.4                                                          10  Compound XIX 5 hr.                                                                        13.5                                                          11  post dose   13.6                                                          12              13.3                                                          13  Compound XI 0.5 hr.                                                                       term. term.      18.0                                         14  post dose   term. term.      17.7                                         15              term. term.      32.4                                         16  Compound XI 2 hr.                                                                         term. 45.7       14.0                                         17  post dose   17.2                                                          18              14.7                                                          19  Compound XI 5 hr.                                                                         13.1                                                          20  post dose   11.4                                                          21              11.5                                                          __________________________________________________________________________

What is claimed is:
 1. A process for preparing compounds of Formulas 3a'and 3b' ##STR26## wherein Q and T can be the same or different aminoprotecting groups, p is 1 or 2, R¹ is phenyl or alkyl substitutedphenyl, n is 1 to 3; comprising the steps of:(a) forming a trianion of acompound of Formula iv using a strong base ##STR27## wherein T, Q and nare as defined above and R² refers to alkyl; (b) reacting the trianionof a compound of Formula iv with a silylimine of Formula v ##STR28##wherein R¹ and p are as defined above and R³, R⁴ and R⁵ canindependently be alkyl, phenyl or alkyl substituted phenyl to afford acompound of Formula vi ##STR29## in which T, Q, n, p, R¹, R⁴, R³ and R⁵are as defined previously; (c) hydrolyzing the nitrogen silicone bond ofa compound of Formula vi to afford a compound of the formula 3, and##STR30## (d) separating the cis and trans isomers of a compound ofFormula 3 to produce compounds of Formulas 3a' and 3b'.
 2. The processof claim 1 in which T and Q are each independently t-butoxycarbonyl orphenylmethoxycarbonyl; n is 1 or 2; p is 1; R¹ is phenyl, R² is methyl;and R³, R⁴ and R⁵ are methyl.